diff --git a/paper-releases/pbisc-diffusion/updates/20261002T124820Z-crosscohort-liu-default/diffusion/dataset/server_1/dependency/2927095b6a41fbcd/pairs_primary.csv b/paper-releases/pbisc-diffusion/updates/20261002T124820Z-crosscohort-liu-default/diffusion/dataset/server_1/dependency/2927095b6a41fbcd/pairs_primary.csv new file mode 100644 index 0000000000000000000000000000000000000000..46d8fe722cfd4d8b54d8a74512245a9071db9d18 --- /dev/null +++ b/paper-releases/pbisc-diffusion/updates/20261002T124820Z-crosscohort-liu-default/diffusion/dataset/server_1/dependency/2927095b6a41fbcd/pairs_primary.csv @@ -0,0 +1,66 @@ +sc_sample,Batch,Donor,Subject,Timepoint,Class,Age,Gender,Ward,Status,symptom_type,hospital_days,day_from_symptom_onset_to_hospitalization,number_of_days_hospitalized,computed_ethnicity,ever_admitted_to_icu,severity,days_of_symptoms_onset,condition,outcome,DDimer,CRP,material_type,sample_bsi_id,sample_alt_id,sample_label,batch,days_since_hospitalized,days_since_onset,severity.outcome,PC1,PC1_cat,cond_group,onset_group,intubation_vent,PC1_onset_group,sample_name,Sorted,BatchSort,n_cells_object,n_sorted_dropped,n_unsorted,n_unsorted_hto_singlet,n_unsorted_hto_doublet,n_unsorted_hto_negative,n_unsorted_not_demuxlet_singlet,n_selected,hashtags,kind,bulk_column,table_file,column_key,column_correction,subject,column_timepoint,bulk_gsm,bulk_title,geo_batch,geo_timepoint_characteristic,geo_timepoint,timepoint,timepoint_conflict,kind_bulk,id_match,sample_id +B1_AA220014_HC,B1,AA220014,AA220014,HC,HC,67,Male,Healthy_control,na,HC,0,,,,,,0,,,,,,,,,,,,,,,HC,,,NA_NA,AA220014_HC,N,B1UnSort,2665,2212,453,428,25,0,0,428,HTO1,HC,AA220014,B1_bulkRNAseq_countmtx.csv,AA220014,,AA220014,,GSM4929233,Batch 1 bulkRNAseq AA220014,B1,Healthy Control,HC,HC,,HC,exact_subject_timepoint,AA220014_HC +B1_AA220534_HC,B1,AA220534,AA220534,HC,HC,47,Male,Healthy_control,na,HC,0,,,,,,0,,,,,,,,,,,,,,,HC,,,NA_NA,AA220534_HC,N,B1UnSort,2000,1630,370,353,17,0,0,353,HTO2,HC,AA220534,B1_bulkRNAseq_countmtx.csv,AA220534,,AA220534,,GSM4929234,Batch1 bulkRNAseq AA220534,B1,Healthy Control,HC,HC,,HC,exact_subject_timepoint,AA220534_HC +B1_AA220907_HC,B1,AA220907,AA220907,HC,HC,32,Female,Healthy_control,na,HC,0,,,,,,0,,,,,,,,,,,,,,,HC,,,NA_NA,AA220907_HC,N,B1UnSort,1820,1418,402,384,18,0,0,384,HTO2,HC,AA220907,B1_bulkRNAseq_countmtx.csv,AA220907,,AA220907,,GSM4929235,Batch 1 bulk RNAseq AA220907,B1,Healthy Control,HC,HC,,HC,exact_subject_timepoint,AA220907_HC +B1_HGR0000071_T0,B1,HGR0000071,HGR0000071,T0,COVID,71,Female,Infectious_Diseases,Deceased,phenotype,,6,,EUR,FALSE,Critical,8,COVID19,deceased,5250,309,PBMC,AA500490 0001,BS-EQ-0005:T0:PBMC,BS-EQ-0005,Batch1,2,8,Critical-Deceased,3.56953465111741,PC1_high,Critical,early,,PC1_high_early,HGR0000071_T0,N,B1UnSort,2510,2143,367,347,20,0,0,347,HTO1,COVID,HGR0000071_T0,B1_bulkRNAseq_countmtx.csv,HGR0000071_T0,,HGR0000071,T0,GSM4929237,Batch 1 bulk RNAseq HGR0000071,B1,Timepoint 0,T0,T0,,COVID,exact_subject_timepoint,HGR0000071_T0 +B1_HGR0000074_T0,B1,HGR0000074,HGR0000074,T0,COVID,52,Male,Infectious_Diseases,Alive,Comorbid,19,9,19,EUR,FALSE,Critical,18,COVID19,alive,2087,1.1,PBMC,AA500491 0001,BS-EQ-0026:T0:PBMC,BS-EQ-0026,Batch1,9,18,Critical-Alive,-0.626780069068832,PC1_low,Critical,mid,,PC1_low_mid,HGR0000074_T0,N,B1UnSort,1796,1433,363,347,16,0,0,347,HTO2,COVID,PHGR0000074_T0,B1_bulkRNAseq_countmtx.csv,HGR0000074_T0,leading 'P' removed (typo; GEO title and HTO metadata name the subject without it),HGR0000074,T0,GSM4929238,Batch 1 bulk RNAseq HGR0000074,B1,Timepoint 0,T0,T0,,COVID,subject_timepoint; bulk column name corrected,HGR0000074_T0 +B1_HGR0000078_T0,B1,HGR0000078,HGR0000078,T0,COVID,40,Female,Tropical_Diseases,Alive,phenotype,21,5,21,EUR,TRUE,Critical,18,COVID19,alive,2242,4.9,PBMC,AA500492 0001,BS-EQ-0003:T0:PBMC,BS-EQ-0003,Batch1,13,18,Critical-Alive,-0.0923749337182958,PC1_high,Critical,mid,TRUE,PC1_high_mid,HGR0000078_T0,N,B1UnSort,899,691,208,186,22,0,0,186,HTO2,COVID,HGR0000078_T0,B1_bulkRNAseq_countmtx.csv,HGR0000078_T0,,HGR0000078,T0,GSM4929239,Batch 1 bulk RNAseq HGR0000078_T0,B1,Timepoint 0,T0,T0,,COVID,exact_subject_timepoint,HGR0000078_T0 +B1_HGR0000078_T1,B1,HGR0000078,HGR0000078,T1,COVID,40,Female,Tropical_Diseases,Alive,phenotype,21,5,21,EUR,TRUE,Critical,23,COVID19,alive,2242,4.9,PBMC,AA500517 0001,BS-EQ-0003:T1:PBMC,BS-EQ-0003,Batch1,18,23,Critical-Alive,-0.0923749337182958,PC1_high,Critical,mid,TRUE,PC1_high_mid,HGR0000078_T1,N,B1UnSort,1125,908,217,204,13,0,0,204,HTO1,COVID,HGR0000078_T1,B1_bulkRNAseq_countmtx.csv,HGR0000078_T1,,HGR0000078,T1,GSM4929240,Batch 1 bulk RNAseq HGR0000078_T1,B1,Timepoint 1,T1,T1,,COVID,exact_subject_timepoint,HGR0000078_T1 +B1_HGR0000079_T0,B1,HGR0000079,HGR0000079,T0,COVID,37,Female,Infectious_Diseases,Alive,phenotype,27,24,27,EUR,FALSE,Critical,30,COVID19-Negative,alive,854,96.2,PBMC,AA500493 0001,BS-EQ-0006:T0:PBMC,BS-EQ-0006,Batch1,6,30,,,,Critical,late,,NA_late,HGR0000079_T0,N,B1UnSort,1405,1282,123,119,4,0,0,119,HTO2,COVID,HGR0000079_T0,B1_bulkRNAseq_countmtx.csv,HGR0000079_T0,,HGR0000079,T0,GSM4929241,Batch 1 bulk RNAseq HGR0000079_T0,B1,Timepoint 0,T0,T0,,COVID,exact_subject_timepoint,HGR0000079_T0 +B1_HGR0000079_T1,B1,HGR0000079,HGR0000079,T1,COVID,37,Female,Infectious_Diseases,Alive,phenotype,27,24,27,EUR,FALSE,Critical,36,COVID19-Negative,alive,854,96.2,PBMC,AA500518 0001,BS-EQ-0006:T1:PBMC,BS-EQ-0006,Batch1,12,36,,,,Critical,late,,NA_late,HGR0000079_T1,N,B1UnSort,955,799,156,145,11,0,0,145,HTO1,COVID,HGR0000079_T1,B1_bulkRNAseq_countmtx.csv,HGR0000079_T1,,HGR0000079,T1,GSM4929242,Batch 1 bulk RNAseq HGR0000079_T1,B1,Timepoint 1,T1,T1,,COVID,exact_subject_timepoint,HGR0000079_T1 +B1_HGR0000083_T0,B1,HGR0000083,HGR0000083,T0,COVID,55,Male,Tropical_Diseases,Alive,phenotype,11,5,11,EUR,FALSE,Moderate,6,COVID19,alive,589,124.9,PBMC,AA500494 0001,BS-EQ-0002:T0:PBMC,BS-EQ-0002,Batch1,1,6,Moderate-Alive,-1.9508583519753,PC1_low,Moderate,early,,PC1_low_early,HGR0000083_T0,N,B1UnSort,2240,2063,177,165,12,0,0,165,HTO2,COVID,HGR0000083_T0,B1_bulkRNAseq_countmtx.csv,HGR0000083_T0,,HGR0000083,T0,GSM4929243,Batch 1 bulk RNAseq HGR0000083_T0,B1,Timepoint 0,T0,T0,,COVID,exact_subject_timepoint,HGR0000083_T0 +B1_HGR0000083_T1,B1,HGR0000083,HGR0000083,T1,COVID,55,Male,Tropical_Diseases,Alive,phenotype,11,5,11,EUR,FALSE,Moderate,13,COVID19,alive,589,124.9,PBMC,AA500519 0001,BS-EQ-0002:T1:PBMC,BS-EQ-0002,Batch1,8,13,Moderate-Alive,-1.9508583519753,PC1_low,Moderate,early,,PC1_low_early,HGR0000083_T1,N,B1UnSort,1680,1365,315,304,11,0,0,304,HTO1,COVID,HGR0000083_T1,B1_bulkRNAseq_countmtx.csv,HGR0000083_T1,,HGR0000083,T1,GSM4929244,Batch 1 bulk RNAseq HGR0000083_T1,B1,Timepoint 1,T1,T1,,COVID,exact_subject_timepoint,HGR0000083_T1 +B1_HGR0000089_T1,B1,HGR0000089,HGR0000089,T1,COVID,78,Male,Infectious_Diseases,Alive,phenotype,14,12,14,EUR,FALSE,Critical,20,COVID19-Negative,alive,217,19.1,PBMC,AA500520 0001,BS-EQ-0027:T1:PBMC,BS-EQ-0027,Batch1,8,20,Critical-Alive,0.0516202369908761,PC1_high,Critical,mid,,PC1_high_mid,HGR0000089_T1,N,B1UnSort,2476,2102,374,365,9,0,0,365,HTO1,COVID,HGR0000089_T1,B1_bulkRNAseq_countmtx.csv,HGR0000089_T1,,HGR0000089,T1,GSM4929245,Batch 1 bulk RNAseq HGR0000089,B1,Timepoint 0,T0,T1,column name T1 vs GEO characteristic 'Timepoint 0',COVID,subject_timepoint; GEO bulk timepoint characteristic differs,HGR0000089_T1 +B1_HGR0000091_T0,B1,HGR0000091,HGR0000091,T0,COVID,35,Male,Infectious_Diseases,Alive,phenotype,5,17,5,EUR,FALSE,Moderate,20,COVID19,alive,511,73.3,PBMC,AA500496 0001,BS-EQ-0004:T0:PBMC,BS-EQ-0004,Batch1,3,20,Moderate-Alive,-1.41813091808461,PC1_low,Moderate,mid,,PC1_low_mid,HGR0000091_T0,N,B1UnSort,1764,1383,381,368,13,0,0,368,HTO1,COVID,HGR0000091_T0,B1_bulkRNAseq_countmtx.csv,HGR0000091_T0,,HGR0000091,T0,GSM4929246,Batch 1 bulk RNAseq HGR0000091,B1,Timepoint 0,T0,T0,,COVID,exact_subject_timepoint,HGR0000091_T0 +B1_HGR0000093_T0,B1,HGR0000093,HGR0000093,T0,COVID,70,Male,Infectious_Diseases,Alive,phenotype,20,10,20,EUR,FALSE,Critical,11,COVID19,alive,327,1.5,PBMC,AA500498 0001,BS-EQ-0008:T0:PBMC,BS-EQ-0008,Batch1,1,11,Critical-Alive,-0.348888393393188,PC1_low,Critical,early,,PC1_low_early,HGR0000093_T0,N,B1UnSort,1773,1288,485,457,28,0,0,457,HTO2,COVID,HGR0000093_T0,B1_bulkRNAseq_countmtx.csv,HGR0000093_T0,,HGR0000093,T0,GSM4929247,Batch 1 bulk RNAseq HGR0000093_T0,B1,Timepoint 0,T0,T0,,COVID,exact_subject_timepoint,HGR0000093_T0 +B1_HGR0000093_T1,B1,HGR0000093,HGR0000093,T1,COVID,70,Male,Infectious_Diseases,Alive,phenotype,20,10,20,EUR,FALSE,Critical,17,COVID19,alive,327,1.5,PBMC,AA500521 0001,BS-EQ-0008:T1:PBMC,BS-EQ-0008,Batch1,7,17,Critical-Alive,-0.348888393393188,PC1_low,Critical,mid,,PC1_low_mid,HGR0000093_T1,N,B1UnSort,981,812,169,154,15,0,0,154,HTO1,COVID,HGR0000093_T1,B1_bulkRNAseq_countmtx.csv,HGR0000093_T1,,HGR0000093,T1,GSM4929248,Batch 1 bulk RNAseq HGR0000093_T1,B1,Timepoint 1,T1,T1,,COVID,exact_subject_timepoint,HGR0000093_T1 +B1_HGR0000098_T0,B1,HGR0000098,HGR0000098,T0,COVID,60,Male,Infectious_Diseases,Alive,phenotype,42,,42,EUR,TRUE,Critical,,COVID19,alive,347,5.6,PBMC,AA500500 0001,BS-EQ-0009:T0:PBMC,BS-EQ-0009,Batch1,1,,Critical-Alive,2.95435018758758,PC1_high,Critical,,TRUE,PC1_high_NA,HGR0000098_T0,N,B1UnSort,2552,2128,424,407,17,0,0,407,HTO1,COVID,HGR0000098_T0,B1_bulkRNAseq_countmtx.csv,HGR0000098_T0,,HGR0000098,T0,GSM4929249,Batch 1 bulk RNAseq HGR0000098,B1,Timepoint 0,T0,T0,,COVID,exact_subject_timepoint,HGR0000098_T0 +B1_HGR0000110_T0,B1,HGR0000110,HGR0000110,T0,COVID,73,Male,Infectious_Diseases,Alive,phenotype,,3,,EUR,FALSE,Critical,3,COVID19,deceased,537,216,PBMC,AA500503 0001,BS-EQ-0013:T0:PBMC,BS-EQ-0013,Batch1,0,3,Critical-Deceased,3.68918247500143,PC1_high,Critical,early,,PC1_high_early,HGR0000110_T0,N,B1UnSort,561,427,134,129,5,0,0,129,HTO2,COVID,HGR0000110_T0,B1_bulkRNAseq_countmtx.csv,HGR0000110_T0,,HGR0000110,T0,GSM4929250,Batch 1 bulk RNAseq HGR0000110,B1,Timepoint 0,T0,T0,,COVID,exact_subject_timepoint,HGR0000110_T0 +B1_HGR0000136_T0,B1,HGR0000136,HGR0000136,T0,COVID,57,Female,Infectious_Diseases,Alive,Comorbid,3,16,3,EUR,FALSE,Moderate,17,COVID19,alive,193,10,PBMC,AA500507 0001,BS-EQ-0019:T0:PBMC,BS-EQ-0019,Batch1,1,17,Moderate-Alive,-2.91073693427368,PC1_low,Moderate,mid,,PC1_low_mid,HGR0000136_T0,N,B1UnSort,1956,1626,330,320,10,0,0,320,HTO1,COVID,HGR0000136_T0,B1_bulkRNAseq_countmtx.csv,HGR0000136_T0,,HGR0000136,T0,GSM4929251,Batch 1 bulk RNAseq HGR0000136,B1,Timepoint 0,T0,T0,,COVID,exact_subject_timepoint,HGR0000136_T0 +B1_HGR0000137_T0,B1,HGR0000137,HGR0000137,T0,COVID,79,Female,Infectious_Diseases,Alive,phenotype,3,3,3,EUR,FALSE,Severe,4,COVID19,alive,363,36.5,PBMC,AA500508 0001,BS-EQ-0020:T0:PBMC,BS-EQ-0020,Batch1,1,4,Severe-Alive,-1.2015610057303,PC1_low,Severe,early,,PC1_low_early,HGR0000137_T0,N,B1UnSort,1980,1635,345,329,16,0,0,329,HTO1,COVID,HGR0000137_T0,B1_bulkRNAseq_countmtx.csv,HGR0000137_T0,,HGR0000137,T0,GSM4929252,Batch 1 bulk RNAseq HGR0000137,B1,Timepoint 0,T0,T0,,COVID,exact_subject_timepoint,HGR0000137_T0 +B1_HGR0000141_T0,B1,HGR0000141,HGR0000141,T0,COVID,76,Male,Infectious_Diseases,Deceased,phenotype,,14,,EUR,FALSE,Critical,20,COVID19,deceased,0,294.8,PBMC,AA500509 0001,BS-EQ-0022:T0:PBMC,BS-EQ-0022,Batch1,6,20,Critical-Deceased,3.27623326424634,PC1_high,Critical,mid,,PC1_high_mid,HGR0000141_T0,N,B1UnSort,1807,1534,273,258,15,0,0,258,HTO2,COVID,HGR0000141_T0,B1_bulkRNAseq_countmtx.csv,HGR0000141_T0,,HGR0000141,T0,GSM4929253,Batch 1 bulk RNAseq HGR0000141,B1,Timepoint 0,T0,T0,,COVID,exact_subject_timepoint,HGR0000141_T0 +B1_HGR0000143_T0,B1,HGR0000143,HGR0000143,T0,COVID,54,Female,Infectious_Diseases,Alive,phenotype,34,7,34,EAS,TRUE,Critical,8,COVID19,alive,0,2.3,PBMC,AA500511 0001,BS-EQ-0024:T0:PBMC,BS-EQ-0024,Batch1,1,8,Critical-Alive,1.67405631685952,PC1_high,Critical,early,TRUE,PC1_high_early,HGR0000143_T0,N,B1UnSort,1636,1336,300,289,11,0,0,289,HTO2,COVID,HGR0000143_T0,B1_bulkRNAseq_countmtx.csv,HGR0000143_T0,,HGR0000143,T0,GSM4929254,Batch 1 bulk RNAseq HGR0000143,B1,Timepoint 0,T0,T0,,COVID,exact_subject_timepoint,HGR0000143_T0 +B1_IM000040_HC,B1,IM000040,IM000040,HC,HC,37,Male,Healthy_control,na,HC,0,,,,,,0,,,,,,,,,,,,,,,HC,,,NA_NA,IM000040_HC,N,B1UnSort,2352,1958,394,379,15,0,0,379,HTO1,HC,IM000040,B1_bulkRNAseq_countmtx.csv,IM000040,,IM000040,,GSM4929255,Batch 1 bulk RNAseq IM000040,B1,Healthy Control,HC,HC,,HC,exact_subject_timepoint,IM000040_HC +B2_HGR0000042_T0,B2,HGR0000042,HGR0000042,T0,COVID,53,Male,Infectious_Diseases,Alive,phenotype,15,5,15,EUR,FALSE,Critical,12,COVID19,alive,0,33,PBMC,AA500488 0001,BS-EQ-0015:T0:PBMC,BS-EQ-0015,Batch2,7,12,Critical-Alive,-0.0144850947759859,PC1_high,Critical,early,,PC1_high_early,HGR0000042_T0,N,B2UnSort,4551,1141,3410,2306,5,1099,0,2306,HTO3,COVID,HGR0000042_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000042_T0,,HGR0000042,T0,GSM4929261,Batch 2 bulk RNAseq HGR0000042_T0,B2,Timepoint 0,T0,T0,,COVID,exact_subject_timepoint,HGR0000042_T0 +B2_HGR0000042_T1,B2,HGR0000042,HGR0000042,T1,COVID,53,Male,Infectious_Diseases,Alive,phenotype,15,5,15,EUR,FALSE,Critical,18,COVID19,alive,0,33,PBMC,AA500530 0001,BS-EQ-0015:T1:PBMC,BS-EQ-0015,Batch2,13,18,Critical-Alive,-0.0144850947759859,PC1_high,Critical,mid,,PC1_high_mid,HGR0000042_T1,N,B2UnSort,6641,1944,4697,4663,2,32,0,4663,HTO2,COVID,HGR0000042_T1,B2_B3_bulkRNAseq_countmtx.csv,HGR0000042_T1,,HGR0000042,T1,GSM4929262,Batch 2 bulk RNAseq HGR0000042_T1,B2,Timepoint 1,T1,T1,,COVID,exact_subject_timepoint,HGR0000042_T1 +B2_HGR0000069_T0,B2,HGR0000069,HGR0000069,T0,COVID,54,Male,Infectious_Diseases,Alive,phenotype,10,12,10,EUR,FALSE,Severe,13,COVID19,alive,593,0.8,PBMC,AA500514 0001,BS-EQ-0001:T0:PBMC,BS-EQ-0001,Batch2,1,13,Critical-Alive,1.04716869546426,PC1_high,Severe,early,,PC1_high_early,HGR0000069_T0,N,B2UnSort,6909,2351,4558,4524,3,31,0,4524,HTO2,COVID,HGR0000069_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000069_T0,,HGR0000069,T0,GSM4929263,Batch 2 bulk RNAseq HGR0000069_T0,B2,Timepoint 0,T0,T0,,COVID,exact_subject_timepoint,HGR0000069_T0 +B2_HGR0000069_T1,B2,HGR0000069,HGR0000069,T1,COVID,54,Male,Infectious_Diseases,Alive,phenotype,10,12,10,EUR,FALSE,Severe,20,COVID19,alive,593,0.8,PBMC,AA500516 0001,BS-EQ-0001:T1:PBMC,BS-EQ-0001,Batch2,8,20,Critical-Alive,1.04716869546426,PC1_high,Severe,mid,,PC1_high_mid,HGR0000069_T1,N,B2UnSort,6981,1534,5447,4670,3,774,0,4670,HTO1,COVID,HGR0000069_T1,B2_B3_bulkRNAseq_countmtx.csv,HGR0000069_T1,,HGR0000069,T1,GSM4929264,Batch 2 bulk RNAseq HGR0000069_T1,B2,Timepoint 1,T1,T1,,COVID,exact_subject_timepoint,HGR0000069_T1 +B2_HGR0000069_T2,B2,HGR0000069,HGR0000069,T2,COVID,54,Male,Infectious_Diseases,Alive,phenotype,10,12,10,EUR,FALSE,Severe,33,COVID19,alive,593,0.8,PBMC,AA500552 0001,BS-EQ-0001:T2:PBMC,BS-EQ-0001,Batch2,21,33,Critical-Alive,1.04716869546426,PC1_high,Severe,late,,PC1_high_late,HGR0000069_T2,N,B2UnSort,5944,1658,4286,2413,7,1866,0,2413,HTO3,COVID,HGR0000069_T2,B2_B3_bulkRNAseq_countmtx.csv,HGR0000069_T2,,HGR0000069,T2,GSM4929265,Batch 2 bulk RNAseq HGR0000069_T2,B2,Timepoint 2,T2,T2,,COVID,exact_subject_timepoint,HGR0000069_T2 +B2_HGR0000092_T0,B2,HGR0000092,HGR0000092,T0,COVID,45,Male,Infectious_Diseases,Alive,phenotype,15,9,15,EUR,FALSE,Critical,11,COVID19,alive,871,179.7,PBMC,AA500497 0001,BS-EQ-0012:T0:PBMC,BS-EQ-0012,Batch2,2,11,Critical-Alive,-0.52272790656358,PC1_low,Critical,early,,PC1_low_early,HGR0000092_T0,N,B2UnSort,7743,2134,5609,5402,9,198,0,5402,HTO1,COVID,HGR0000092_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000092_T0,,HGR0000092,T0,GSM4929266,Batch 2 bulk RNAseq HGR0000092_T0,B2,Timepoint 0,T0,T0,,COVID,exact_subject_timepoint,HGR0000092_T0 +B2_HGR0000092_T1,B2,HGR0000092,HGR0000092,T1,COVID,45,Male,Infectious_Diseases,Alive,phenotype,15,9,15,EUR,FALSE,Critical,18,COVID19,alive,871,179.7,PBMC,AA500531 0001,BS-EQ-0012:T1:PBMC,BS-EQ-0012,Batch2,9,18,Critical-Alive,-0.52272790656358,PC1_low,Critical,mid,,PC1_low_mid,HGR0000092_T1,N,B2UnSort,6429,1938,4491,3015,9,1467,0,3015,HTO3,COVID,HGR0000092_T1,B2_B3_bulkRNAseq_countmtx.csv,HGR0000092_T1,,HGR0000092,T1,GSM4929267,Batch 2 bulk RNAseq HGR0000092_T1,B2,Timepoint 1,T1,T1,,COVID,exact_subject_timepoint,HGR0000092_T1 +B2_HGR0000094_T0,B2,HGR0000094,HGR0000094,T0,COVID,61,Male,Infectious_Diseases,Alive,Comorbid,14,5,14,EUR,FALSE,Critical,6,COVID19-Negative,alive,382,0,PBMC,AA500499 0001,BS-EQ-0007:T0:PBMC,BS-EQ-0007,Batch2,1,6,Critical-Alive,0.726261730620933,PC1_high,Critical,early,,PC1_high_early,HGR0000094_T0,N,B2UnSort,4591,1418,3173,3148,2,23,0,3148,HTO2,COVID,HGR0000094_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000094_T0,,HGR0000094,T0,GSM4929268,Batch 2 bulk RNAseq HGR0000094_T0,B2,Timepoint 0,T0,T0,,COVID,exact_subject_timepoint,HGR0000094_T0 +B2_HGR0000094_T1,B2,HGR0000094,HGR0000094,T1,COVID,61,Male,Infectious_Diseases,Alive,Comorbid,14,5,14,EUR,FALSE,Critical,13,COVID19-Negative,alive,382,0,PBMC,AA500532 0001,BS-EQ-0007:T1:PBMC,BS-EQ-0007,Batch2,8,13,Critical-Alive,0.726261730620933,PC1_high,Critical,early,,PC1_high_early,HGR0000094_T1,N,B2UnSort,2554,671,1883,967,2,914,0,967,HTO3,COVID,HGR0000094_T1,B2_B3_bulkRNAseq_countmtx.csv,HGR0000094_T1,,HGR0000094,T1,GSM4929269,Batch 2 bulk RNAseq HGR0000094_T1,B2,Timepoint 1,T1,T1,,COVID,exact_subject_timepoint,HGR0000094_T1 +B2_HGR0000115_T1,B2,HGR0000115,HGR0000115,T1,COVID,24,Male,Infectious_Diseases,Alive,phenotype,9,3,9,EUR,FALSE,Severe,11,COVID19,alive,0,14.8,PBMC,AA500535 0001,BS-EQ-0032:T1:PBMC,BS-EQ-0032,Batch2,8,11,Severe-Alive,-2.76779602430212,PC1_low,Severe,early,,PC1_low_early,HGR0000115_T1,N,B2UnSort,8138,2823,5315,5285,1,29,0,5285,HTO2,COVID,HGR0000115_T1,B2_B3_bulkRNAseq_countmtx.csv,HGR0000115_T1,,HGR0000115,T1,GSM4929270,Batch 2 bulk RNAseq HGR0000115_T1,B2,Timepoint 1,T1,T1,,COVID,exact_subject_timepoint,HGR0000115_T1 +B2_HGR0000124_T0,B2,HGR0000124,HGR0000124,T0,COVID,79,Male,Infectious_Diseases,Alive,phenotype,44,8,44,EUR,FALSE,Critical,36,COVID19,alive,0,0,PBMC,AA500504 0001,BS-EQ-0021:T0:PBMC,BS-EQ-0021,Batch2,28,36,,,,Critical,late,,NA_late,HGR0000124_T0,N,B2UnSort,14056,4842,9214,9135,6,73,0,9135,HTO2,COVID,HGR0000124_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000124_T0,,HGR0000124,T0,GSM4929271,Batch 2 bulk RNAseq HGR0000124_T0,B2,Timepoint 0,T0,T0,,COVID,exact_subject_timepoint,HGR0000124_T0 +B2_HGR0000124_T1,B2,HGR0000124,HGR0000124,T1,COVID,79,Male,Infectious_Diseases,Alive,phenotype,44,8,44,EUR,FALSE,Critical,49,COVID19,alive,0,0,PBMC,AA500537 0001,BS-EQ-0021:T1:PBMC,BS-EQ-0021,Batch2,41,49,,,,Critical,late,,NA_late,HGR0000124_T1,N,B2UnSort,5969,1928,4041,2617,1,1423,0,2617,HTO3,COVID,HGR0000124_T1,B2_B3_bulkRNAseq_countmtx.csv,HGR0000124_T1,,HGR0000124,T1,GSM4929272,Batch 2 bulk RNAseq HGR0000124_T1,B2,Timepoint 1,T1,T1,,COVID,exact_subject_timepoint,HGR0000124_T1 +B2_HGR0000144_T0,B2,HGR0000144,HGR0000144,T0,COVID,84,Female,Infectious_Diseases,Alive,phenotype,25,12,25,EUR,FALSE,Severe,15,COVID19,alive,872,179,PBMC,AA500512 0001,BS-EQ-0028:T0:PBMC,BS-EQ-0028,Batch2,3,15,Severe-Alive,-0.158291992685593,PC1_high,Severe,early,,PC1_high_early,HGR0000144_T0,N,B2UnSort,5288,999,4289,2283,5,2001,0,2283,HTO3,COVID,HGR0000144_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000144_T0,,HGR0000144,T0,GSM4929273,Batch 2 bulk RNAseq HGR0000144_T0,B2,Timepoint 0,T0,T0,,COVID,exact_subject_timepoint,HGR0000144_T0 +B2_HGR0000144_T2,B2,HGR0000144,HGR0000144,T2,COVID,84,Female,Infectious_Diseases,Alive,phenotype,25,12,25,EUR,FALSE,Severe,28,COVID19,alive,872,179,PBMC,AA500559 0001,BS-EQ-0028:T2:PBMC,BS-EQ-0028,Batch2,16,28,Severe-Alive,-0.158291992685593,PC1_high,Severe,late,,PC1_high_late,HGR0000144_T2,N,B2UnSort,8949,1691,7258,6434,8,816,0,6434,HTO1,COVID,HGR0000144_T2,B2_B3_bulkRNAseq_countmtx.csv,HGR0000144_T2,,HGR0000144,T2,GSM4929274,Batch 2 bulk RNAseq HGR0000144_T2,B2,Timepoint 2,T2,T2,,COVID,exact_subject_timepoint,HGR0000144_T2 +B2_HGR0000391_T0,B2,HGR0000391,HGR0000391,T0,COVID,66,Male,Infectious_Diseases,Alive,Comorbid,9,5,9,EUR,FALSE,Critical,6,COVID19,alive,220,2.2,PBMC,AA500523 0001,BS-EQ-0029:T0:PBMC,BS-EQ-0029,Batch2,1,6,Critical-Alive,-1.30858460669219,PC1_low,Critical,early,,PC1_low_early,HGR0000391_T0,N,B2UnSort,9292,2382,6910,6846,5,59,0,6846,HTO1,COVID,HGR0000391_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000391_T0,,HGR0000391,T0,GSM4929275,Batch 2 bulk RNAseq HGR0000391_T0,B2,Timepoint 0,T0,T0,,COVID,exact_subject_timepoint,HGR0000391_T0 +B2_HGR0000395_T0,B2,HGR0000395,HGR0000395,T0,COVID,50,Male,Tropical_Diseases,Alive,phenotype,8,12,8,EUR,FALSE,Severe,14,COVID19,alive,229,12.1,PBMC,AA500525 0001,BS-EQ-0034:T0:PBMC,BS-EQ-0034,Batch2,2,14,Severe-Alive,-2.51313825051465,PC1_low,Severe,early,,PC1_low_early,HGR0000395_T0,N,B2UnSort,5628,1557,4071,2871,3,1197,0,2871,HTO3,COVID,HGR0000395_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000395_T0,,HGR0000395,T0,GSM4929276,Batch 2 bulk RNAseq HGR0000395_T0,B2,Timepoint 0,T0,T0,,COVID,exact_subject_timepoint,HGR0000395_T0 +B2_HGR0000418_T1,B2,HGR0000418,HGR0000418,T1,COVID,50,Male,Infectious_Diseases,Alive,phenotype,14,21,14,EUR,FALSE,Critical,32,COVID19,alive,0,95.4,PBMC,AA500546 0001,BS-EQ-0036:T1:PBMC,BS-EQ-0036,Batch2,11,32,,,,Critical,late,,NA_late,HGR0000418_T1,N,B2UnSort,8474,1828,6646,6313,5,328,0,6313,HTO1,COVID,HGR0000418_T1,B2_B3_bulkRNAseq_countmtx.csv,HGR0000418_T1,,HGR0000418,T1,GSM4929277,Batch 2 bulk RNAseq HGR0000418_T1,B2,Timepoint 1,T1,T1,,COVID,exact_subject_timepoint,HGR0000418_T1 +B2_SHD2_HC,B2,SHD2,SHD2,HC,HC,53,Male,Healthy_control,na,HC,0,,,,,,0,,,,,,,,,,,,,,,HC,,,NA_NA,SHD2_HC,N,B2UnSort,8603,2885,5718,3538,1,2179,0,3538,HTO3,HC,SHD2,B2_B3_bulkRNAseq_countmtx.csv,SHD2,,SHD2,,GSM4929278,Batch 2 bulk RNAseq SHD2,B2,Healthy Control,HC,HC,,HC,exact_subject_timepoint,SHD2_HC +B2_SHD4_HC,B2,SHD4,SHD4,HC,HC,63,Male,Healthy_control,na,HC,0,,,,,,0,,,,,,,,,,,,,,,HC,,,NA_NA,SHD4_HC,N,B2UnSort,10177,2890,7287,7240,7,40,0,7240,HTO1,HC,SHD4,B2_B3_bulkRNAseq_countmtx.csv,SHD4,,SHD4,,GSM4929279,Batch 2 bulk RNAseq SHD4,B2,Healthy Control,HC,HC,,HC,exact_subject_timepoint,SHD4_HC +B2_SHD7_HC,B2,SHD7,SHD7,HC,HC,77,Male,Healthy_control,na,HC,0,,,,,,0,,,,,,,,,,,,,,,HC,,,NA_NA,SHD7_HC,N,B2UnSort,9502,2989,6513,6488,2,23,0,6488,HTO2,HC,SHD7,B2_B3_bulkRNAseq_countmtx.csv,SHD7,,SHD7,,GSM4929280,Batch 2 bulk RNAseq SHD7,B2,Healthy Control,HC,HC,,HC,exact_subject_timepoint,SHD7_HC +B3_HGR0000051_T0,B3,HGR0000051,HGR0000051,T0,COVID,55,Male,Tropical_Diseases,Alive,phenotype,14,7,14,EUR,FALSE,Critical,10,COVID19,alive,618,276.8,PBMC,AA500489 0001,BS-EQ-0014:T0:PBMC,BS-EQ-0014,Batch3,3,10,Critical-Alive,0.0824050089906214,PC1_high,Critical,early,,PC1_high_early,HGR0000051_T0,N,B3UnSort,4378,1153,3225,3158,67,0,0,3158,HTO3,COVID,HGR0000051_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000051_T0,,HGR0000051,T0,GSM4929283,Batch 3 bulk RNAseq HGR0000051_T0,B3,Timepoint 0,T0,T0,,COVID,exact_subject_timepoint,HGR0000051_T0 +B3_HGR0000051_T1,B3,HGR0000051,HGR0000051,T1,COVID,55,Male,Tropical_Diseases,Alive,phenotype,14,7,14,EUR,FALSE,Critical,14,COVID19,alive,618,276.8,PBMC,AA500515 0001,BS-EQ-0014:T1:PBMC,BS-EQ-0014,Batch3,7,14,Critical-Alive,0.0824050089906214,PC1_high,Critical,early,,PC1_high_early,HGR0000051_T1,N,B3UnSort,6772,1715,5057,5057,0,0,0,5057,HTO2,COVID,HGR0000051_T1,B2_B3_bulkRNAseq_countmtx.csv,HGR0000051_T1,,HGR0000051,T1,GSM4929284,Batch 3 bulk RNAseq HGR0000051_T1,B3,Timepoint 1,T1,T1,,COVID,exact_subject_timepoint,HGR0000051_T1 +B3_HGR0000051_T2,B3,HGR0000051,HGR0000051,T2,COVID,55,Male,Tropical_Diseases,Alive,phenotype,14,7,14,EUR,FALSE,Critical,17,COVID19,alive,618,276.8,PBMC,AA500551 0001,BS-EQ-0014:T2:PBMC,BS-EQ-0014,Batch3,10,17,Critical-Alive,0.0824050089906214,PC1_high,Critical,mid,,PC1_high_mid,HGR0000051_T2,N,B3UnSort,6060,1773,4287,2179,71,2037,0,2179,HTO5,COVID,HGR0000051_T2,B2_B3_bulkRNAseq_countmtx.csv,HGR0000051_T2,,HGR0000051,T2,GSM4929285,Batch 3 bulk RNAseq HGR0000051_T2,B3,Timepoint 2,T2,T2,,COVID,exact_subject_timepoint,HGR0000051_T2 +B3_HGR0000051_T3,B3,HGR0000051,HGR0000051,T3,COVID,55,Male,Tropical_Diseases,Alive,phenotype,14,7,14,EUR,FALSE,Critical,20,COVID19,alive,618,276.8,PBMC,AA500563 0001,BS-EQ-0014:T3:PBMC,BS-EQ-0014,Batch3,13,20,Critical-Alive,0.0824050089906214,PC1_high,Critical,mid,,PC1_high_mid,HGR0000051_T3,N,B3UnSort,7400,2207,5193,5119,74,0,0,5119,HTO1,COVID,HGR0000051_T3,B2_B3_bulkRNAseq_countmtx.csv,HGR0000051_T3,,HGR0000051,T3,GSM4929286,Batch 3 bulk RNAseq HGR0000051_T3,B3,Timepoint 3,T3,T3,,COVID,exact_subject_timepoint,HGR0000051_T3 +B3_HGR0000101_T0,B3,HGR0000101,HGR0000101,T0,COVID,73,Male,Tropical_Diseases,Alive,phenotype,22,7,22,EUR,FALSE,Critical,8,COVID19,alive,5211,68,PBMC,AA500501 0001,BS-EQ-0011:T0:PBMC,BS-EQ-0011,Batch3,1,8,Critical-Alive,2.38816634675649,PC1_high,Critical,early,,PC1_high_early,HGR0000101_T0,N,B3UnSort,2904,890,2014,977,23,1014,0,977,HTO5,COVID,HGR0000101_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000101_T0,,HGR0000101,T0,GSM4929287,Batch 3 bulk RNAseq HGR0000101_T0,B3,Timepoint 0,T0,T0,,COVID,exact_subject_timepoint,HGR0000101_T0 +B3_HGR0000101_T1,B3,HGR0000101,HGR0000101,T1,COVID,73,Male,Tropical_Diseases,Alive,phenotype,22,7,22,EUR,FALSE,Critical,14,COVID19,alive,5211,68,PBMC,AA500522 0001,BS-EQ-0011:T1:PBMC,BS-EQ-0011,Batch3,7,14,Critical-Alive,2.38816634675649,PC1_high,Critical,early,,PC1_high_early,HGR0000101_T1,N,B3UnSort,7872,2801,5071,5010,61,0,0,5010,HTO1,COVID,HGR0000101_T1,B2_B3_bulkRNAseq_countmtx.csv,HGR0000101_T1,,HGR0000101,T1,GSM4929288,Batch 3 bulk RNAseq HGR0000101_T1,B3,Timepoint 1,T1,T1,,COVID,exact_subject_timepoint,HGR0000101_T1 +B3_HGR0000101_T3,B3,HGR0000101,HGR0000101,T3,COVID,73,Male,Tropical_Diseases,Alive,phenotype,22,7,22,EUR,FALSE,Critical,27,COVID19,alive,5211,68,PBMC,AA500564 0001,BS-EQ-0011:T3:PBMC,BS-EQ-0011,Batch3,20,27,Critical-Alive,2.38816634675649,PC1_high,Critical,late,,PC1_high_late,HGR0000101_T3,N,B3UnSort,6075,1999,4076,4001,75,0,0,4001,HTO3,COVID,HGR0000101_T3,B2_B3_bulkRNAseq_countmtx.csv,HGR0000101_T3,,HGR0000101,T3,GSM4929289,Batch 3 bulk RNAseq HGR0000101_T3,B3,Timepoint 3,T3,T3,,COVID,exact_subject_timepoint,HGR0000101_T3 +B3_HGR0000102_T0,B3,HGR0000102,HGR0000102,T0,COVID,58,Male,Infectious_Diseases,Alive,phenotype,24,7,24,EUR,FALSE,Critical,10,COVID19,alive,208,4.9,PBMC,AA500502 0001,BS-EQ-0010:T0:PBMC,BS-EQ-0010,Batch3,3,10,Critical-Alive,-1.57139207217568,PC1_low,Critical,early,,PC1_low_early,HGR0000102_T0,N,B3UnSort,7555,2860,4695,4626,69,0,0,4626,HTO3,COVID,HGR0000102_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000102_T0,,HGR0000102,T0,GSM4929290,Batch 3 bulk RNAseq HGR0000102_T0,B3,Timepoint 0,T0,T0,,COVID,exact_subject_timepoint,HGR0000102_T0 +B3_HGR0000102_T1,B3,HGR0000102,HGR0000102,T1,COVID,58,Male,Infectious_Diseases,Alive,phenotype,24,7,24,EUR,FALSE,Critical,17,COVID19,alive,208,4.9,PBMC,AA500533 0001,BS-EQ-0010:T1:PBMC,BS-EQ-0010,Batch3,10,17,Critical-Alive,-1.57139207217568,PC1_low,Critical,mid,,PC1_low_mid,HGR0000102_T1,N,B3UnSort,9292,3508,5784,5690,94,0,0,5690,HTO1,COVID,HGR0000102_T1,B2_B3_bulkRNAseq_countmtx.csv,HGR0000102_T1,,HGR0000102,T1,GSM4929291,Batch 3 bulk RNAseq HGR0000102_T1,B3,Timepoint 1,T1,T1,,COVID,exact_subject_timepoint,HGR0000102_T1 +B3_HGR0000134_T0,B3,HGR0000134,HGR0000134,T0,COVID,53,Male,Tropical_Diseases,Alive,Comorbid,8,14,8,EUR,FALSE,Critical,16,COVID19,alive,0,19.3,PBMC,AA500505 0001,BS-EQ-0017:T0:PBMC,BS-EQ-0017,Batch3,2,16,Critical-Alive,-1.92544770891706,PC1_low,Critical,early,,PC1_low_early,HGR0000134_T0,N,B3UnSort,8210,2121,6089,6089,0,0,0,6089,HTO2,COVID,HGR0000134_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000134_T0,,HGR0000134,T0,GSM4929292,Batch 3 bulk RNAseq HGR0000134_T0,B3,Timepoint 0,T0,T0,,COVID,exact_subject_timepoint,HGR0000134_T0 +B3_HGR0000134_T1,B3,HGR0000134,HGR0000134,T1,COVID,53,Male,Tropical_Diseases,Alive,Comorbid,8,14,8,EUR,FALSE,Critical,22,COVID19,alive,0,19.3,PBMC,AA500538 0001,BS-EQ-0017:T1:PBMC,BS-EQ-0017,Batch3,8,22,Critical-Alive,-1.92544770891706,PC1_low,Critical,mid,,PC1_low_mid,HGR0000134_T1,N,B3UnSort,9372,2918,6454,2783,84,3587,0,2783,HTO5,COVID,HGR0000134_T1,B2_B3_bulkRNAseq_countmtx.csv,HGR0000134_T1,,HGR0000134,T1,GSM4929293,Batch 3 bulk RNAseq HGR0000134_T1,B3,Timepoint 1,T1,T1,,COVID,exact_subject_timepoint,HGR0000134_T1 +B3_HGR0000135_T0,B3,HGR0000135,HGR0000135,T0,COVID,59,Male,Infectious_Diseases,Alive,phenotype,14,7,14,EUR,FALSE,Critical,10,COVID19,alive,0,30.7,PBMC,AA500506 0001,BS-EQ-0018:T0:PBMC,BS-EQ-0018,Batch3,3,10,Critical-Alive,0.75305315128394,PC1_high,Critical,early,,PC1_high_early,HGR0000135_T0,N,B3UnSort,8841,2772,6069,6069,0,0,0,6069,HTO2,COVID,HGR0000135_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000135_T0,,HGR0000135,T0,GSM4929294,Batch 3 bulk RNAseq HGR0000135_T0,B3,Timepoint 0,T0,T0,,COVID,exact_subject_timepoint,HGR0000135_T0 +B3_HGR0000135_T1,B3,HGR0000135,HGR0000135,T1,COVID,59,Male,Infectious_Diseases,Alive,phenotype,14,7,14,EUR,FALSE,Critical,16,COVID19,alive,0,30.7,PBMC,AA500539 0001,BS-EQ-0018:T1:PBMC,BS-EQ-0018,Batch3,9,16,Critical-Alive,0.75305315128394,PC1_high,Critical,early,,PC1_high_early,HGR0000135_T1,N,B3UnSort,7509,2380,5129,5019,110,0,0,5019,HTO3,COVID,HGR0000135_T1,B2_B3_bulkRNAseq_countmtx.csv,HGR0000135_T1,,HGR0000135,T1,GSM4929295,Batch 3 bulk RNAseq HGR0000135_T1,B3,Timepoint 1,T1,T1,,COVID,exact_subject_timepoint,HGR0000135_T1 +B3_HGR0000142_T0,B3,HGR0000142,HGR0000142,T0,COVID,49,Male,Infectious_Diseases,Alive,Comorbid,9,12,9,EUR,FALSE,Critical,12,COVID19,alive,291,16.1,PBMC,AA500510 0001,BS-EQ-0023:T0:PBMC,BS-EQ-0023,Batch3,0,12,Critical-Alive,-0.617734576041256,PC1_low,Critical,early,,PC1_low_early,HGR0000142_T0,N,B3UnSort,7893,2761,5132,2866,80,2186,0,2866,HTO5,COVID,HGR0000142_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000142_T0,,HGR0000142,T0,GSM4929296,Batch 3 bulk RNAseq HGR0000142_T0,B3,Timepoint 0,T0,T0,,COVID,exact_subject_timepoint,HGR0000142_T0 +B3_HGR0000392_T0,B3,HGR0000392,HGR0000392,T0,COVID,52,Male,Infectious_Diseases,Alive,phenotype,14,0,14,UNKNOWN,FALSE,Critical,2,COVID19,alive,0,0,PBMC,AA500524 0001,BS-EQ-0033:T0:PBMC,BS-EQ-0033,Batch3,2,2,Critical-Alive,-1.84274100791149,PC1_low,Critical,early,,PC1_low_early,HGR0000392_T0,N,B3UnSort,8061,2650,5411,5355,56,0,0,5355,HTO1,COVID,HGR0000392_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000392_T0,,HGR0000392,T0,GSM4929297,Batch 3 bulk RNAseq HGR0000392_T0,B3,Timepoint 0,T0,T0,,COVID,exact_subject_timepoint,HGR0000392_T0 +B3_HGR0000392_T3,B3,HGR0000392,HGR0000392,T3,COVID,52,Male,Infectious_Diseases,Alive,phenotype,14,0,14,UNKNOWN,FALSE,Critical,13,COVID19,alive,0,0,PBMC,AA500565 0001,BS-EQ-0033:T3:PBMC,BS-EQ-0033,Batch3,13,13,Critical-Alive,-1.84274100791149,PC1_low,Critical,early,,PC1_low_early,HGR0000392_T3,N,B3UnSort,7958,3106,4852,1445,62,3345,0,1445,HTO5,COVID,HGR0000392_T3,B2_B3_bulkRNAseq_countmtx.csv,HGR0000392_T3,,HGR0000392,T3,GSM4929298,Batch 3 bulk RNAseq HGR0000392_T3,B3,Timepoint 3,T3,T3,,COVID,exact_subject_timepoint,HGR0000392_T3 +B3_HGR0000429_T1,B3,HGR0000429,HGR0000429,T1,COVID,54,Male,Infectious_Diseases,Alive,phenotype,13,16,13,,FALSE,Severe,26,COVID19,alive,0,124.8,PBMC,AA500549 0001,BS-EQ-0043:T1:PBMC,BS-EQ-0043,Batch3,10,26,Severe-Alive,-1.18921567127169,PC1_low,Severe,late,,PC1_low_late,HGR0000429_T1,N,B3UnSort,10783,3377,7406,7406,0,0,0,7406,HTO2,COVID,HGR0000429_T1,B2_B3_bulkRNAseq_countmtx.csv,HGR0000429_T1,,HGR0000429,T1,GSM4929299,Batch 3 bulk RNAseq HGR0000429_T1,B3,Timepoint 1,T1,T1,,COVID,exact_subject_timepoint,HGR0000429_T1 +B3_HGR0000429_T2,B3,HGR0000429,HGR0000429,T2,COVID,54,Male,Infectious_Diseases,Alive,phenotype,13,16,13,,FALSE,Severe,28,COVID19,alive,0,124.8,PBMC,AA500562 0001,BS-EQ-0043:T2:PBMC,BS-EQ-0043,Batch3,12,28,Severe-Alive,-1.18921567127169,PC1_low,Severe,late,,PC1_low_late,HGR0000429_T2,N,B3UnSort,8031,2940,5091,5000,91,0,0,5000,HTO3,COVID,HGR0000429_T2,B2_B3_bulkRNAseq_countmtx.csv,HGR0000429_T2,,HGR0000429,T2,GSM4929300,Batch 3 bulk RNAseq HGR0000429_T2,B3,Timepoint 2,T2,T2,,COVID,exact_subject_timepoint,HGR0000429_T2 +B3_HGR0000430_T1,B3,HGR0000430,HGR0000430,T1,COVID,77,Female,Tropical_Diseases,Alive,Comorbid,,7,,,FALSE,Critical,20,COVID19,deceased,0,63.5,PBMC,AA500550 0001,BS-EQ-0044:T1:PBMC,BS-EQ-0044,Batch3,13,20,Critical-Deceased,2.76885345317608,PC1_high,Critical,mid,,PC1_high_mid,HGR0000430_T1,N,B3UnSort,5262,1814,3448,1941,73,1434,0,1941,HTO5,COVID,HGR0000430_T1,B2_B3_bulkRNAseq_countmtx.csv,HGR0000430_T1,,HGR0000430,T1,GSM4929301,Batch 3 bulk RNAseq HGR0000430_T1,B3,Timepoint 1,T1,T1,,COVID,exact_subject_timepoint,HGR0000430_T1 +B3_SHD1_HC,B3,SHD1,SHD1,HC,HC,51,Male,Healthy_control,na,HC,0,,,,,,0,,,,,,,,,,,,,,,HC,,,NA_NA,SHD1_HC,N,B3UnSort,9623,3373,6250,6250,0,0,0,6250,HTO2,HC,SHD1,B2_B3_bulkRNAseq_countmtx.csv,SHD1,,SHD1,,GSM4929302,Batch 3 bulk RNAseq SHD1,B3,Healthy Control,HC,HC,,HC,exact_subject_timepoint,SHD1_HC +B3_SHD3_HC,B3,SHD3,SHD3,HC,HC,58,Male,Healthy_control,na,HC,0,,,,,,0,,,,,,,,,,,,,,,HC,,,NA_NA,SHD3_HC,N,B3UnSort,9440,4087,5353,5254,99,0,0,5254,HTO3,HC,SHD3,B2_B3_bulkRNAseq_countmtx.csv,SHD3,,SHD3,,GSM4929303,Batch 3 bulk RNAseq SHD3,B3,Healthy Control,HC,HC,,HC,exact_subject_timepoint,SHD3_HC +B3_SHD5_HC,B3,SHD5,SHD5,HC,HC,73,Male,Healthy_control,na,HC,0,,,,,,0,,,,,,,,,,,,,,,HC,,,NA_NA,SHD5_HC,N,B3UnSort,11962,5358,6604,6524,80,0,0,6524,HTO1,HC,SHD5,B2_B3_bulkRNAseq_countmtx.csv,SHD5,,SHD5,,GSM4929304,Batch 3 bulk RNAseq SHD5,B3,Healthy Control,HC,HC,,HC,exact_subject_timepoint,SHD5_HC +B3_SHD6_HC,B3,SHD6,SHD6,HC,HC,74,Female,Healthy_control,na,HC,0,,,,,,0,,,,,,,,,,,,,,,HC,,,NA_NA,SHD6_HC,N,B3UnSort,9127,3531,5596,2554,70,2972,0,2554,HTO5,HC,SHD6,B2_B3_bulkRNAseq_countmtx.csv,SHD6,,SHD6,,GSM4929305,Batch 3 bulk RNAseq SHD6,B3,Healthy Control,HC,HC,,HC,exact_subject_timepoint,SHD6_HC diff --git a/paper-releases/pbisc-diffusion/updates/20261002T124820Z-crosscohort-liu-default/diffusion/dataset/server_1/dependency/3d2d8d3c278bb125/bulk_count_columns.csv b/paper-releases/pbisc-diffusion/updates/20261002T124820Z-crosscohort-liu-default/diffusion/dataset/server_1/dependency/3d2d8d3c278bb125/bulk_count_columns.csv new file mode 100644 index 0000000000000000000000000000000000000000..019bd3516eb7ecb19fa680880daa2049f3a22b9e --- /dev/null +++ b/paper-releases/pbisc-diffusion/updates/20261002T124820Z-crosscohort-liu-default/diffusion/dataset/server_1/dependency/3d2d8d3c278bb125/bulk_count_columns.csv @@ -0,0 +1,74 @@ +bulk_column,table_file,column_key,column_correction,subject,column_timepoint,bulk_gsm,bulk_title,geo_batch,geo_timepoint_characteristic,geo_timepoint,timepoint,timepoint_conflict,kind,paired,unpaired_reason +HGR0000091_T0,B1_bulkRNAseq_countmtx.csv,HGR0000091_T0,,HGR0000091,T0,GSM4929246,Batch 1 bulk RNAseq HGR0000091,B1,Timepoint 0,T0,T0,,COVID,True, +HGR0000071_T0,B1_bulkRNAseq_countmtx.csv,HGR0000071_T0,,HGR0000071,T0,GSM4929237,Batch 1 bulk RNAseq HGR0000071,B1,Timepoint 0,T0,T0,,COVID,True, +HGR0000098_T0,B1_bulkRNAseq_countmtx.csv,HGR0000098_T0,,HGR0000098,T0,GSM4929249,Batch 1 bulk RNAseq HGR0000098,B1,Timepoint 0,T0,T0,,COVID,True, +HGR0000136_T0,B1_bulkRNAseq_countmtx.csv,HGR0000136_T0,,HGR0000136,T0,GSM4929251,Batch 1 bulk RNAseq HGR0000136,B1,Timepoint 0,T0,T0,,COVID,True, +HGR0000137_T0,B1_bulkRNAseq_countmtx.csv,HGR0000137_T0,,HGR0000137,T0,GSM4929252,Batch 1 bulk RNAseq HGR0000137,B1,Timepoint 0,T0,T0,,COVID,True, +HGR0000089_T1,B1_bulkRNAseq_countmtx.csv,HGR0000089_T1,,HGR0000089,T1,GSM4929245,Batch 1 bulk RNAseq HGR0000089,B1,Timepoint 0,T0,T1,column name T1 vs GEO characteristic 'Timepoint 0',COVID,True, +HGR0000093_T1,B1_bulkRNAseq_countmtx.csv,HGR0000093_T1,,HGR0000093,T1,GSM4929248,Batch 1 bulk RNAseq HGR0000093_T1,B1,Timepoint 1,T1,T1,,COVID,True, +HGR0000083_T1,B1_bulkRNAseq_countmtx.csv,HGR0000083_T1,,HGR0000083,T1,GSM4929244,Batch 1 bulk RNAseq HGR0000083_T1,B1,Timepoint 1,T1,T1,,COVID,True, +HGR0000079_T1,B1_bulkRNAseq_countmtx.csv,HGR0000079_T1,,HGR0000079,T1,GSM4929242,Batch 1 bulk RNAseq HGR0000079_T1,B1,Timepoint 1,T1,T1,,COVID,True, +HGR0000078_T1,B1_bulkRNAseq_countmtx.csv,HGR0000078_T1,,HGR0000078,T1,GSM4929240,Batch 1 bulk RNAseq HGR0000078_T1,B1,Timepoint 1,T1,T1,,COVID,True, +IM000040,B1_bulkRNAseq_countmtx.csv,IM000040,,IM000040,,GSM4929255,Batch 1 bulk RNAseq IM000040,B1,Healthy Control,HC,HC,,HC,True, +AA220014,B1_bulkRNAseq_countmtx.csv,AA220014,,AA220014,,GSM4929233,Batch 1 bulkRNAseq AA220014,B1,Healthy Control,HC,HC,,HC,True, +HGR0000141_T0,B1_bulkRNAseq_countmtx.csv,HGR0000141_T0,,HGR0000141,T0,GSM4929253,Batch 1 bulk RNAseq HGR0000141,B1,Timepoint 0,T0,T0,,COVID,True, +HGR0000143_T0,B1_bulkRNAseq_countmtx.csv,HGR0000143_T0,,HGR0000143,T0,GSM4929254,Batch 1 bulk RNAseq HGR0000143,B1,Timepoint 0,T0,T0,,COVID,True, +PHGR0000074_T0,B1_bulkRNAseq_countmtx.csv,HGR0000074_T0,leading 'P' removed (typo; GEO title and HTO metadata name the subject without it),HGR0000074,T0,GSM4929238,Batch 1 bulk RNAseq HGR0000074,B1,Timepoint 0,T0,T0,,COVID,True, +HGR0000110_T0,B1_bulkRNAseq_countmtx.csv,HGR0000110_T0,,HGR0000110,T0,GSM4929250,Batch 1 bulk RNAseq HGR0000110,B1,Timepoint 0,T0,T0,,COVID,True, +HGR0000093_T0,B1_bulkRNAseq_countmtx.csv,HGR0000093_T0,,HGR0000093,T0,GSM4929247,Batch 1 bulk RNAseq HGR0000093_T0,B1,Timepoint 0,T0,T0,,COVID,True, +HGR0000083_T0,B1_bulkRNAseq_countmtx.csv,HGR0000083_T0,,HGR0000083,T0,GSM4929243,Batch 1 bulk RNAseq HGR0000083_T0,B1,Timepoint 0,T0,T0,,COVID,True, +HGR0000079_T0,B1_bulkRNAseq_countmtx.csv,HGR0000079_T0,,HGR0000079,T0,GSM4929241,Batch 1 bulk RNAseq HGR0000079_T0,B1,Timepoint 0,T0,T0,,COVID,True, +HGR0000078_T0,B1_bulkRNAseq_countmtx.csv,HGR0000078_T0,,HGR0000078,T0,GSM4929239,Batch 1 bulk RNAseq HGR0000078_T0,B1,Timepoint 0,T0,T0,,COVID,True, +AA220534,B1_bulkRNAseq_countmtx.csv,AA220534,,AA220534,,GSM4929234,Batch1 bulkRNAseq AA220534,B1,Healthy Control,HC,HC,,HC,True, +AA220907,B1_bulkRNAseq_countmtx.csv,AA220907,,AA220907,,GSM4929235,Batch 1 bulk RNAseq AA220907,B1,Healthy Control,HC,HC,,HC,True, +CHI-014,B1_bulkRNAseq_countmtx.csv,CHI-014,,CHI-014,,GSM4929236,Batch 1 bulk RNAseq CHI-014,B1,Technical Control,Technical Control,Technical Control,,technical_control,False,technical control column (CHI014 / HD buffy-coat test); excluded by task rule +CHI014_B2,B2_B3_bulkRNAseq_countmtx.csv,CHI014_B2,,CHI014_B2,,GSM4929256,Batch 2 bulk RNAseq CHI014_B2,B2,Healthy Control,HC,HC,,technical_control,False,technical control column (CHI014 / HD buffy-coat test); excluded by task rule +CHI014_B3,B2_B3_bulkRNAseq_countmtx.csv,CHI014_B3,,CHI014_B3,,GSM4929282,Batch 3 bulk RNAseq CHI014_B3,B3,Technical Control,Technical Control,Technical Control,,technical_control,False,technical control column (CHI014 / HD buffy-coat test); excluded by task rule +HD_ML_bc,B2_B3_bulkRNAseq_countmtx.csv,HD_ML_bc,,HD_ML_bc,,GSM4929257,Batch 2 bulk RNAseq HD_ML_bc,B2,Technical test sample,Technical test sample,Technical test sample,,technical_control,False,technical control column (CHI014 / HD buffy-coat test); excluded by task rule +HD_ML_PBMC,B2_B3_bulkRNAseq_countmtx.csv,HD_ML_PBMC,,HD_ML_PBMC,,GSM4929258,Batch 2 bulk RNAseq HD_ML_PBMC,B2,Healthy Control,HC,HC,,technical_control,False,technical control column (CHI014 / HD buffy-coat test); excluded by task rule +HD_VO_bc,B2_B3_bulkRNAseq_countmtx.csv,HD_VO_bc,,HD_VO_bc,,GSM4929259,Batch 2 bulk RNAseq HD_VO_bc,B2,Technical test sample,Technical test sample,Technical test sample,,technical_control,False,technical control column (CHI014 / HD buffy-coat test); excluded by task rule +HD_VO_PBMC,B2_B3_bulkRNAseq_countmtx.csv,HD_VO_PBMC,,HD_VO_PBMC,,GSM4929260,Batch 2 bulk RNAseq HD_VO_PBMC,B2,Healthy Control,HC,HC,,technical_control,False,technical control column (CHI014 / HD buffy-coat test); excluded by task rule +HGR0000042_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000042_T0,,HGR0000042,T0,GSM4929261,Batch 2 bulk RNAseq HGR0000042_T0,B2,Timepoint 0,T0,T0,,COVID,True, +HGR0000042_T1,B2_B3_bulkRNAseq_countmtx.csv,HGR0000042_T1,,HGR0000042,T1,GSM4929262,Batch 2 bulk RNAseq HGR0000042_T1,B2,Timepoint 1,T1,T1,,COVID,True, +HGR0000051_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000051_T0,,HGR0000051,T0,GSM4929283,Batch 3 bulk RNAseq HGR0000051_T0,B3,Timepoint 0,T0,T0,,COVID,True, +HGR0000051_T1,B2_B3_bulkRNAseq_countmtx.csv,HGR0000051_T1,,HGR0000051,T1,GSM4929284,Batch 3 bulk RNAseq HGR0000051_T1,B3,Timepoint 1,T1,T1,,COVID,True, +HGR0000051_T2,B2_B3_bulkRNAseq_countmtx.csv,HGR0000051_T2,,HGR0000051,T2,GSM4929285,Batch 3 bulk RNAseq HGR0000051_T2,B3,Timepoint 2,T2,T2,,COVID,True, +HGR0000051_T3,B2_B3_bulkRNAseq_countmtx.csv,HGR0000051_T3,,HGR0000051,T3,GSM4929286,Batch 3 bulk RNAseq HGR0000051_T3,B3,Timepoint 3,T3,T3,,COVID,True, +HGR0000069_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000069_T0,,HGR0000069,T0,GSM4929263,Batch 2 bulk RNAseq HGR0000069_T0,B2,Timepoint 0,T0,T0,,COVID,True, +HGR0000069_T1,B2_B3_bulkRNAseq_countmtx.csv,HGR0000069_T1,,HGR0000069,T1,GSM4929264,Batch 2 bulk RNAseq HGR0000069_T1,B2,Timepoint 1,T1,T1,,COVID,True, +HGR0000069_T2,B2_B3_bulkRNAseq_countmtx.csv,HGR0000069_T2,,HGR0000069,T2,GSM4929265,Batch 2 bulk RNAseq HGR0000069_T2,B2,Timepoint 2,T2,T2,,COVID,True, +HGR0000092_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000092_T0,,HGR0000092,T0,GSM4929266,Batch 2 bulk RNAseq HGR0000092_T0,B2,Timepoint 0,T0,T0,,COVID,True, +HGR0000092_T1,B2_B3_bulkRNAseq_countmtx.csv,HGR0000092_T1,,HGR0000092,T1,GSM4929267,Batch 2 bulk RNAseq HGR0000092_T1,B2,Timepoint 1,T1,T1,,COVID,True, +HGR0000094_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000094_T0,,HGR0000094,T0,GSM4929268,Batch 2 bulk RNAseq HGR0000094_T0,B2,Timepoint 0,T0,T0,,COVID,True, +HGR0000094_T1,B2_B3_bulkRNAseq_countmtx.csv,HGR0000094_T1,,HGR0000094,T1,GSM4929269,Batch 2 bulk RNAseq HGR0000094_T1,B2,Timepoint 1,T1,T1,,COVID,True, +HGR0000101_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000101_T0,,HGR0000101,T0,GSM4929287,Batch 3 bulk RNAseq HGR0000101_T0,B3,Timepoint 0,T0,T0,,COVID,True, +HGR0000101_T1,B2_B3_bulkRNAseq_countmtx.csv,HGR0000101_T1,,HGR0000101,T1,GSM4929288,Batch 3 bulk RNAseq HGR0000101_T1,B3,Timepoint 1,T1,T1,,COVID,True, +HGR0000101_T3,B2_B3_bulkRNAseq_countmtx.csv,HGR0000101_T3,,HGR0000101,T3,GSM4929289,Batch 3 bulk RNAseq HGR0000101_T3,B3,Timepoint 3,T3,T3,,COVID,True, +HGR0000102_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000102_T0,,HGR0000102,T0,GSM4929290,Batch 3 bulk RNAseq HGR0000102_T0,B3,Timepoint 0,T0,T0,,COVID,True, +HGR0000102_T1,B2_B3_bulkRNAseq_countmtx.csv,HGR0000102_T1,,HGR0000102,T1,GSM4929291,Batch 3 bulk RNAseq HGR0000102_T1,B3,Timepoint 1,T1,T1,,COVID,True, +HGR0000115_T1,B2_B3_bulkRNAseq_countmtx.csv,HGR0000115_T1,,HGR0000115,T1,GSM4929270,Batch 2 bulk RNAseq HGR0000115_T1,B2,Timepoint 1,T1,T1,,COVID,True, +HGR0000124_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000124_T0,,HGR0000124,T0,GSM4929271,Batch 2 bulk RNAseq HGR0000124_T0,B2,Timepoint 0,T0,T0,,COVID,True, +HGR0000124_T1,B2_B3_bulkRNAseq_countmtx.csv,HGR0000124_T1,,HGR0000124,T1,GSM4929272,Batch 2 bulk RNAseq HGR0000124_T1,B2,Timepoint 1,T1,T1,,COVID,True, +HGR0000134_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000134_T0,,HGR0000134,T0,GSM4929292,Batch 3 bulk RNAseq HGR0000134_T0,B3,Timepoint 0,T0,T0,,COVID,True, +HGR0000134_T1,B2_B3_bulkRNAseq_countmtx.csv,HGR0000134_T1,,HGR0000134,T1,GSM4929293,Batch 3 bulk RNAseq HGR0000134_T1,B3,Timepoint 1,T1,T1,,COVID,True, +HGR0000135_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000135_T0,,HGR0000135,T0,GSM4929294,Batch 3 bulk RNAseq HGR0000135_T0,B3,Timepoint 0,T0,T0,,COVID,True, +HGR0000135_T1,B2_B3_bulkRNAseq_countmtx.csv,HGR0000135_T1,,HGR0000135,T1,GSM4929295,Batch 3 bulk RNAseq HGR0000135_T1,B3,Timepoint 1,T1,T1,,COVID,True, +HGR0000142_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000142_T0,,HGR0000142,T0,GSM4929296,Batch 3 bulk RNAseq HGR0000142_T0,B3,Timepoint 0,T0,T0,,COVID,True, +HGR0000144_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000144_T0,,HGR0000144,T0,GSM4929273,Batch 2 bulk RNAseq HGR0000144_T0,B2,Timepoint 0,T0,T0,,COVID,True, +HGR0000144_T2,B2_B3_bulkRNAseq_countmtx.csv,HGR0000144_T2,,HGR0000144,T2,GSM4929274,Batch 2 bulk RNAseq HGR0000144_T2,B2,Timepoint 2,T2,T2,,COVID,True, +HGR0000391_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000391_T0,,HGR0000391,T0,GSM4929275,Batch 2 bulk RNAseq HGR0000391_T0,B2,Timepoint 0,T0,T0,,COVID,True, +HGR0000392_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000392_T0,,HGR0000392,T0,GSM4929297,Batch 3 bulk RNAseq HGR0000392_T0,B3,Timepoint 0,T0,T0,,COVID,True, +HGR0000392_T3,B2_B3_bulkRNAseq_countmtx.csv,HGR0000392_T3,,HGR0000392,T3,GSM4929298,Batch 3 bulk RNAseq HGR0000392_T3,B3,Timepoint 3,T3,T3,,COVID,True, +HGR0000395_T0,B2_B3_bulkRNAseq_countmtx.csv,HGR0000395_T0,,HGR0000395,T0,GSM4929276,Batch 2 bulk RNAseq HGR0000395_T0,B2,Timepoint 0,T0,T0,,COVID,True, +HGR0000418_T1,B2_B3_bulkRNAseq_countmtx.csv,HGR0000418_T1,,HGR0000418,T1,GSM4929277,Batch 2 bulk RNAseq HGR0000418_T1,B2,Timepoint 1,T1,T1,,COVID,True, +HGR0000429_T1,B2_B3_bulkRNAseq_countmtx.csv,HGR0000429_T1,,HGR0000429,T1,GSM4929299,Batch 3 bulk RNAseq HGR0000429_T1,B3,Timepoint 1,T1,T1,,COVID,True, +HGR0000429_T2,B2_B3_bulkRNAseq_countmtx.csv,HGR0000429_T2,,HGR0000429,T2,GSM4929300,Batch 3 bulk RNAseq HGR0000429_T2,B3,Timepoint 2,T2,T2,,COVID,True, +HGR0000430_T1,B2_B3_bulkRNAseq_countmtx.csv,HGR0000430_T1,,HGR0000430,T1,GSM4929301,Batch 3 bulk RNAseq HGR0000430_T1,B3,Timepoint 1,T1,T1,,COVID,True, +SHD1,B2_B3_bulkRNAseq_countmtx.csv,SHD1,,SHD1,,GSM4929302,Batch 3 bulk RNAseq SHD1,B3,Healthy Control,HC,HC,,HC,True, +SHD2,B2_B3_bulkRNAseq_countmtx.csv,SHD2,,SHD2,,GSM4929278,Batch 2 bulk RNAseq SHD2,B2,Healthy Control,HC,HC,,HC,True, +SHD3,B2_B3_bulkRNAseq_countmtx.csv,SHD3,,SHD3,,GSM4929303,Batch 3 bulk RNAseq SHD3,B3,Healthy Control,HC,HC,,HC,True, +SHD4,B2_B3_bulkRNAseq_countmtx.csv,SHD4,,SHD4,,GSM4929279,Batch 2 bulk RNAseq SHD4,B2,Healthy Control,HC,HC,,HC,True, +SHD5,B2_B3_bulkRNAseq_countmtx.csv,SHD5,,SHD5,,GSM4929304,Batch 3 bulk RNAseq SHD5,B3,Healthy Control,HC,HC,,HC,True, +SHD6,B2_B3_bulkRNAseq_countmtx.csv,SHD6,,SHD6,,GSM4929305,Batch 3 bulk RNAseq SHD6,B3,Healthy Control,HC,HC,,HC,True, +SHD7,B2_B3_bulkRNAseq_countmtx.csv,SHD7,,SHD7,,GSM4929280,Batch 2 bulk RNAseq SHD7,B2,Healthy Control,HC,HC,,HC,True, +SHD8,B2_B3_bulkRNAseq_countmtx.csv,SHD8,,SHD8,,GSM4929281,Batch 2 bulk RNAseq SHD8,B2,Healthy Control (excluded due to abnormal lymphoma-like B cells found upon analysis),HC,HC,,author_excluded,False,GEO: 'Healthy Control (excluded due to abnormal lymphoma-like B cells found upon analysis)'; the author Seurat object holds 0 cells of this subject diff --git a/paper-releases/pbisc-diffusion/updates/20261002T124820Z-crosscohort-liu-default/diffusion/dataset/server_1/dependency/8f4f1f9892aefcc2/GSE161918_samples.csv b/paper-releases/pbisc-diffusion/updates/20261002T124820Z-crosscohort-liu-default/diffusion/dataset/server_1/dependency/8f4f1f9892aefcc2/GSE161918_samples.csv new file mode 100644 index 0000000000000000000000000000000000000000..584a59d984df88d1d0ffc1af2d222111e8c6f9a4 --- /dev/null +++ b/paper-releases/pbisc-diffusion/updates/20261002T124820Z-crosscohort-liu-default/diffusion/dataset/server_1/dependency/8f4f1f9892aefcc2/GSE161918_samples.csv @@ -0,0 +1,242 @@ +gsm,Sample_title,Sample_geo_accession,Sample_status,Sample_submission_date,Sample_last_update_date,Sample_type,Sample_channel_count,Sample_source_name_ch1,Sample_organism_ch1,Sample_taxid_ch1,ch_unsorted or sorted t/b cells,ch_timepoint,ch_molecule,Sample_molecule_ch1,Sample_extract_protocol_ch1,Sample_description,Sample_data_processing,Sample_platform_id,Sample_contact_name,Sample_contact_email,Sample_contact_department,Sample_contact_institute,Sample_contact_address,Sample_contact_city,Sample_contact_state,Sample_contact_zip/postal_code,Sample_contact_country,Sample_instrument_model,Sample_library_selection,Sample_library_source,Sample_library_strategy,Sample_relation,Sample_supplementary_file_1,Sample_series_id,Sample_data_row_count +GSM4929065,Batch 1 PBMC 10x Lane 1 CSP,GSM4929065,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted and Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873442|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556520,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929065/suppl/GSM4929065_B1_10xlane1_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929066,Batch 1 PBMC 10x Lane 2 CSP,GSM4929066,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted and Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873441|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556521,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929066/suppl/GSM4929066_B1_10xlane2_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929067,Batch 1 PBMC 10x Lane 3 CSP,GSM4929067,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted and Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873440|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556522,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929067/suppl/GSM4929067_B1_10xlane3_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929068,Batch 1 PBMC 10x Lane 4 CSP,GSM4929068,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted and Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873471|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556523,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929068/suppl/GSM4929068_B1_10xlane4_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929069,Batch 1 PBMC 10x Lane 5 CSP,GSM4929069,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted and Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873470|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556524,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929069/suppl/GSM4929069_B1_10xlane5_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929070,Batch 1 PBMC 10x Lane 6 CSP,GSM4929070,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted and Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873469|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556525,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929070/suppl/GSM4929070_B1_10xlane6_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929071,Batch 1 PBMC 10x Lane 7 CSP,GSM4929071,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted and Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873468|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556526,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929071/suppl/GSM4929071_B1_10xlane7_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929072,Batch 1 PBMC 10x Lane 8 CSP,GSM4929072,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted and Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873467|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556527,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929072/suppl/GSM4929072_B1_10xlane8_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929073,Batch 1 PBMC 10x Lane 1 TCR,GSM4929073,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted and Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B1_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873466|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556528,NONE,GSE161918,0 +GSM4929074,Batch 1 PBMC 10x Lane 2 TCR,GSM4929074,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted and Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B1_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873465|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556529,NONE,GSE161918,0 +GSM4929075,Batch 1 PBMC 10x Lane 3 TCR,GSM4929075,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted and Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B1_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873811|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556530,NONE,GSE161918,0 +GSM4929076,Batch 1 PBMC 10x Lane 4 TCR,GSM4929076,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted and Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B1_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873810|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556531,NONE,GSE161918,0 +GSM4929077,Batch 1 PBMC 10x Lane 5 TCR,GSM4929077,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted and Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B1_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873809|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556532,NONE,GSE161918,0 +GSM4929078,Batch 1 PBMC 10x Lane 6 TCR,GSM4929078,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted and Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B1_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873808|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556533,NONE,GSE161918,0 +GSM4929079,Batch 1 PBMC 10x Lane 7 TCR,GSM4929079,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted and Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B1_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873807|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556534,NONE,GSE161918,0 +GSM4929080,Batch 1 PBMC 10x Lane 8 TCR,GSM4929080,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted and Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B1_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873833|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556535,NONE,GSE161918,0 +GSM4929081,Batch 1 PBMC 10x Lane 1 RNA,GSM4929081,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted and Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873832|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556536,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929081/suppl/GSM4929081_B1_10xlane1_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929082,Batch 1 PBMC 10x Lane 2 RNA,GSM4929082,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted and Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873831|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556537,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929082/suppl/GSM4929082_B1_10xlane2_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929083,Batch 1 PBMC 10x Lane 3 RNA,GSM4929083,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted and Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873830|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556538,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929083/suppl/GSM4929083_B1_10xlane3_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929084,Batch 1 PBMC 10x Lane 4 RNA,GSM4929084,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted and Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873829|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556539,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929084/suppl/GSM4929084_B1_10xlane4_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929085,Batch 1 PBMC 10x Lane 5 RNA,GSM4929085,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted and Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873828|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556540,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929085/suppl/GSM4929085_B1_10xlane5_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929086,Batch 1 PBMC 10x Lane 6 RNA,GSM4929086,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted and Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873827|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556541,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929086/suppl/GSM4929086_B1_10xlane6_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929087,Batch 1 PBMC 10x Lane 7 RNA,GSM4929087,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted and Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873826|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556542,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929087/suppl/GSM4929087_B1_10xlane7_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929088,Batch 1 PBMC 10x Lane 8 RNA,GSM4929088,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted and Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873825|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556543,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929088/suppl/GSM4929088_B1_10xlane8_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929089,Batch 2 PBMC 10x Lane 1 CSP,GSM4929089,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873819|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556544,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929089/suppl/GSM4929089_B2_10xlane01_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929090,Batch 2 PBMC 10x Lane 2 CSP,GSM4929090,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873818|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556545,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929090/suppl/GSM4929090_B2_10xlane02_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929091,Batch 2 PBMC 10x Lane 3 CSP,GSM4929091,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873824|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556546,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929091/suppl/GSM4929091_B2_10xlane03_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929092,Batch 2 PBMC 10x Lane 4 CSP,GSM4929092,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873823|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556547,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929092/suppl/GSM4929092_B2_10xlane04_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929093,Batch 2 PBMC 10x Lane 5 CSP,GSM4929093,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873822|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556548,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929093/suppl/GSM4929093_B2_10xlane05_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929094,Batch 2 PBMC 10x Lane 6 CSP,GSM4929094,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873821|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556549,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929094/suppl/GSM4929094_B2_10xlane06_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929095,Batch 2 PBMC 10x Lane 7 CSP,GSM4929095,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873820|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556550,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929095/suppl/GSM4929095_B2_10xlane07_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929096,Batch 2 PBMC 10x Lane 8 CSP,GSM4929096,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873817|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556551,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929096/suppl/GSM4929096_B2_10xlane08_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929097,Batch 2 PBMC 10x Lane 9 CSP,GSM4929097,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873853|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556552,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929097/suppl/GSM4929097_B2_10xlane09_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929098,Batch 2 PBMC 10x Lane 10 CSP,GSM4929098,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873852|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556553,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929098/suppl/GSM4929098_B2_10xlane10_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929099,Batch 2 PBMC 10x Lane 11 CSP,GSM4929099,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873851|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556554,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929099/suppl/GSM4929099_B2_10xlane11_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929100,Batch 2 PBMC 10x Lane 12 CSP,GSM4929100,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873850|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556555,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929100/suppl/GSM4929100_B2_10xlane12_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929101,Batch 2 PBMC 10x Lane 13 CSP,GSM4929101,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873849|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556556,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929101/suppl/GSM4929101_B2_10xlane13_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929102,Batch 2 PBMC 10x Lane 14 CSP,GSM4929102,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873848|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556557,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929102/suppl/GSM4929102_B2_10xlane14_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929103,Batch 2 PBMC 10x Lane 15 CSP,GSM4929103,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873847|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556558,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929103/suppl/GSM4929103_B2_10xlane15_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929104,Batch 2 PBMC 10x Lane 16 CSP,GSM4929104,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873846|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556559,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929104/suppl/GSM4929104_B2_10xlane16_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929105,Batch 2 PBMC 10x Lane 17 CSP,GSM4929105,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873845|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556560,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929105/suppl/GSM4929105_B2_10xlane17_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929106,Batch 2 PBMC 10x Lane 18 CSP,GSM4929106,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873844|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556561,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929106/suppl/GSM4929106_B2_10xlane18_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929107,Batch 2 PBMC 10x Lane 19 CSP,GSM4929107,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873843|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556562,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929107/suppl/GSM4929107_B2_10xlane19_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929108,Batch 2 PBMC 10x Lane 20 CSP,GSM4929108,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873842|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556563,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929108/suppl/GSM4929108_B2_10xlane20_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929109,Batch 2 PBMC 10x Lane 21 CSP,GSM4929109,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873841|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556564,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929109/suppl/GSM4929109_B2_10xlane21_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929110,Batch 2 PBMC 10x Lane 22 CSP,GSM4929110,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873840|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556565,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929110/suppl/GSM4929110_B2_10xlane22_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929111,Batch 2 PBMC 10x Lane 23 CSP,GSM4929111,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873839|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556566,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929111/suppl/GSM4929111_B2_10xlane23_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929112,Batch 2 PBMC 10x Lane 24 CSP,GSM4929112,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873838|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556567,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929112/suppl/GSM4929112_B2_10xlane24_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929113,Batch 2 PBMC 10x Lane 1 TCR,GSM4929113,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B2_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873837|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556568,NONE,GSE161918,0 +GSM4929114,Batch 2 PBMC 10x Lane 2 TCR,GSM4929114,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B2_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873836|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556569,NONE,GSE161918,0 +GSM4929115,Batch 2 PBMC 10x Lane 3 TCR,GSM4929115,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B2_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873835|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556570,NONE,GSE161918,0 +GSM4929116,Batch 2 PBMC 10x Lane 4 TCR,GSM4929116,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B2_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873834|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556571,NONE,GSE161918,0 +GSM4929117,Batch 2 PBMC 10x Lane 5 TCR,GSM4929117,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B2_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873872|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556572,NONE,GSE161918,0 +GSM4929118,Batch 2 PBMC 10x Lane 6 TCR,GSM4929118,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B2_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873871|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556573,NONE,GSE161918,0 +GSM4929119,Batch 2 PBMC 10x Lane 7 TCR,GSM4929119,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B2_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873870|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556574,NONE,GSE161918,0 +GSM4929120,Batch 2 PBMC 10x Lane 8 TCR,GSM4929120,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B2_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873867|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556575,NONE,GSE161918,0 +GSM4929121,Batch 2 PBMC 10x Lane 9 TCR,GSM4929121,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B2_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873866|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556576,NONE,GSE161918,0 +GSM4929122,Batch 2 PBMC 10x Lane 10 TCR,GSM4929122,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B2_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873865|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556577,NONE,GSE161918,0 +GSM4929123,Batch 2 PBMC 10x Lane 11 TCR,GSM4929123,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B2_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873864|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556578,NONE,GSE161918,0 +GSM4929124,Batch 2 PBMC 10x Lane 12 TCR,GSM4929124,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B2_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873863|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556579,NONE,GSE161918,0 +GSM4929125,Batch 2 PBMC 10x Lane 13 TCR,GSM4929125,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B2_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873869|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556580,NONE,GSE161918,0 +GSM4929126,Batch 2 PBMC 10x Lane 14 TCR,GSM4929126,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B2_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873868|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556581,NONE,GSE161918,0 +GSM4929127,Batch 2 PBMC 10x Lane 15 TCR,GSM4929127,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B2_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873862|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556582,NONE,GSE161918,0 +GSM4929128,Batch 2 PBMC 10x Lane 16 TCR,GSM4929128,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B2_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873861|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556583,NONE,GSE161918,0 +GSM4929129,Batch 2 PBMC 10x Lane 17 TCR,GSM4929129,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B2_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873860|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556584,NONE,GSE161918,0 +GSM4929130,Batch 2 PBMC 10x Lane 18 TCR,GSM4929130,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B2_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873859|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556585,NONE,GSE161918,0 +GSM4929131,Batch 2 PBMC 10x Lane 19 TCR,GSM4929131,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B2_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873858|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556586,NONE,GSE161918,0 +GSM4929132,Batch 2 PBMC 10x Lane 20 TCR,GSM4929132,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B2_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873857|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556587,NONE,GSE161918,0 +GSM4929133,Batch 2 PBMC 10x Lane 21 TCR,GSM4929133,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B2_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873856|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556588,NONE,GSE161918,0 +GSM4929134,Batch 2 PBMC 10x Lane 22 TCR,GSM4929134,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B2_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873464|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556589,NONE,GSE161918,0 +GSM4929135,Batch 2 PBMC 10x Lane 23 TCR,GSM4929135,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B2_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873463|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556590,NONE,GSE161918,0 +GSM4929136,Batch 2 PBMC 10x Lane 24 TCR,GSM4929136,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B2_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873462|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556591,NONE,GSE161918,0 +GSM4929137,Batch 2 PBMC 10x Lane 1 RNA,GSM4929137,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873461|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556592,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929137/suppl/GSM4929137_B2_10xlane01_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929138,Batch 2 PBMC 10x Lane 2 RNA,GSM4929138,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873460|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556593,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929138/suppl/GSM4929138_B2_10xlane02_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929139,Batch 2 PBMC 10x Lane 3 RNA,GSM4929139,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873459|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556594,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929139/suppl/GSM4929139_B2_10xlane03_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929140,Batch 2 PBMC 10x Lane 4 RNA,GSM4929140,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873458|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556595,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929140/suppl/GSM4929140_B2_10xlane04_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929141,Batch 2 PBMC 10x Lane 5 RNA,GSM4929141,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873480|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556596,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929141/suppl/GSM4929141_B2_10xlane05_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929142,Batch 2 PBMC 10x Lane 6 RNA,GSM4929142,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873457|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556597,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929142/suppl/GSM4929142_B2_10xlane06_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929143,Batch 2 PBMC 10x Lane 7 RNA,GSM4929143,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873485|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556598,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929143/suppl/GSM4929143_B2_10xlane07_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929144,Batch 2 PBMC 10x Lane 8 RNA,GSM4929144,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873484|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556599,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929144/suppl/GSM4929144_B2_10xlane08_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929145,Batch 2 PBMC 10x Lane 9 RNA,GSM4929145,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873483|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556600,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929145/suppl/GSM4929145_B2_10xlane09_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929146,Batch 2 PBMC 10x Lane 10 RNA,GSM4929146,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873482|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556601,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929146/suppl/GSM4929146_B2_10xlane10_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929147,Batch 2 PBMC 10x Lane 11 RNA,GSM4929147,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873481|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556602,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929147/suppl/GSM4929147_B2_10xlane11_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929148,Batch 2 PBMC 10x Lane 12 RNA,GSM4929148,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873479|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556603,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929148/suppl/GSM4929148_B2_10xlane12_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929149,Batch 2 PBMC 10x Lane 13 RNA,GSM4929149,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873478|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556604,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929149/suppl/GSM4929149_B2_10xlane13_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929150,Batch 2 PBMC 10x Lane 14 RNA,GSM4929150,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873477|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556605,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929150/suppl/GSM4929150_B2_10xlane14_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929151,Batch 2 PBMC 10x Lane 15 RNA,GSM4929151,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873476|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556606,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929151/suppl/GSM4929151_B2_10xlane15_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929152,Batch 2 PBMC 10x Lane 16 RNA,GSM4929152,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873475|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556607,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929152/suppl/GSM4929152_B2_10xlane16_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929153,Batch 2 PBMC 10x Lane 17 RNA,GSM4929153,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873474|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556608,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929153/suppl/GSM4929153_B2_10xlane17_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929154,Batch 2 PBMC 10x Lane 18 RNA,GSM4929154,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873473|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556609,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929154/suppl/GSM4929154_B2_10xlane18_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929155,Batch 2 PBMC 10x Lane 19 RNA,GSM4929155,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873472|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556610,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929155/suppl/GSM4929155_B2_10xlane19_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929156,Batch 2 PBMC 10x Lane 20 RNA,GSM4929156,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873789|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556611,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929156/suppl/GSM4929156_B2_10xlane20_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929157,Batch 2 PBMC 10x Lane 21 RNA,GSM4929157,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873788|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556612,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929157/suppl/GSM4929157_B2_10xlane21_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929158,Batch 2 PBMC 10x Lane 22 RNA,GSM4929158,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873787|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556613,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929158/suppl/GSM4929158_B2_10xlane22_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929159,Batch 2 PBMC 10x Lane 23 RNA,GSM4929159,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873786|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556614,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929159/suppl/GSM4929159_B2_10xlane23_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929160,Batch 2 PBMC 10x Lane 24 RNA,GSM4929160,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873785|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556615,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929160/suppl/GSM4929160_B2_10xlane24_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929161,Batch 3 PBMC 10x Lane 1 CSP,GSM4929161,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873784|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556616,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929161/suppl/GSM4929161_B3_10xlane01_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929162,Batch 3 PBMC 10x Lane 2 CSP,GSM4929162,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873783|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556617,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929162/suppl/GSM4929162_B3_10xlane02_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929163,Batch 3 PBMC 10x Lane 3 CSP,GSM4929163,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873782|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556618,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929163/suppl/GSM4929163_B3_10xlane03_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929164,Batch 3 PBMC 10x Lane 4 CSP,GSM4929164,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873781|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556619,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929164/suppl/GSM4929164_B3_10xlane04_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929165,Batch 3 PBMC 10x Lane 5 CSP,GSM4929165,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873780|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556620,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929165/suppl/GSM4929165_B3_10xlane05_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929166,Batch 3 PBMC 10x Lane 6 CSP,GSM4929166,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873779|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556621,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929166/suppl/GSM4929166_B3_10xlane06_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929167,Batch 3 PBMC 10x Lane 7 CSP,GSM4929167,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873778|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556622,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929167/suppl/GSM4929167_B3_10xlane07_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929168,Batch 3 PBMC 10x Lane 8 CSP,GSM4929168,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873489|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556623,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929168/suppl/GSM4929168_B3_10xlane08_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929169,Batch 3 PBMC 10x Lane 9 CSP,GSM4929169,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873488|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556624,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929169/suppl/GSM4929169_B3_10xlane09_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929170,Batch 3 PBMC 10x Lane 10 CSP,GSM4929170,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873487|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556625,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929170/suppl/GSM4929170_B3_10xlane10_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929171,Batch 3 PBMC 10x Lane 11 CSP,GSM4929171,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873486|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556626,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929171/suppl/GSM4929171_B3_10xlane11_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929172,Batch 3 PBMC 10x Lane 12 CSP,GSM4929172,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873806|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556627,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929172/suppl/GSM4929172_B3_10xlane12_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929173,Batch 3 PBMC 10x Lane 13 CSP,GSM4929173,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873805|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556628,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929173/suppl/GSM4929173_B3_10xlane13_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929174,Batch 3 PBMC 10x Lane 14 CSP,GSM4929174,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873804|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556629,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929174/suppl/GSM4929174_B3_10xlane14_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929175,Batch 3 PBMC 10x Lane 15 CSP,GSM4929175,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873803|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556630,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929175/suppl/GSM4929175_B3_10xlane15_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929176,Batch 3 PBMC 10x Lane 16 CSP,GSM4929176,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873802|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556631,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929176/suppl/GSM4929176_B3_10xlane16_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929177,Batch 3 PBMC 10x Lane 17 CSP,GSM4929177,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873801|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556632,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929177/suppl/GSM4929177_B3_10xlane17_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929178,Batch 3 PBMC 10x Lane 18 CSP,GSM4929178,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873793|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556633,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929178/suppl/GSM4929178_B3_10xlane18_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929179,Batch 3 PBMC 10x Lane 19 CSP,GSM4929179,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873800|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556634,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929179/suppl/GSM4929179_B3_10xlane19_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929180,Batch 3 PBMC 10x Lane 20 CSP,GSM4929180,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873799|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556635,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929180/suppl/GSM4929180_B3_10xlane20_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929181,Batch 3 PBMC 10x Lane 21 CSP,GSM4929181,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873798|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556636,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929181/suppl/GSM4929181_B3_10xlane21_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929182,Batch 3 PBMC 10x Lane 22 CSP,GSM4929182,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873797|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556637,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929182/suppl/GSM4929182_B3_10xlane22_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929183,Batch 3 PBMC 10x Lane 23 CSP,GSM4929183,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873796|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556638,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929183/suppl/GSM4929183_B3_10xlane23_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929184,Batch 3 PBMC 10x Lane 24 CSP,GSM4929184,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,Cell Surface Protein Tag reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873795|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556639,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929184/suppl/GSM4929184_B3_10xlane24_CSP_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929185,Batch 3 PBMC 10x Lane 1 TCR,GSM4929185,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B3_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873794|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556640,NONE,GSE161918,0 +GSM4929186,Batch 3 PBMC 10x Lane 2 TCR,GSM4929186,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B3_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873792|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556641,NONE,GSE161918,0 +GSM4929187,Batch 3 PBMC 10x Lane 3 TCR,GSM4929187,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B3_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873791|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556642,NONE,GSE161918,0 +GSM4929188,Batch 3 PBMC 10x Lane 4 TCR,GSM4929188,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B3_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873790|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556643,NONE,GSE161918,0 +GSM4929189,Batch 3 PBMC 10x Lane 5 TCR,GSM4929189,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B3_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873816|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556644,NONE,GSE161918,0 +GSM4929190,Batch 3 PBMC 10x Lane 6 TCR,GSM4929190,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B3_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873815|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556645,NONE,GSE161918,0 +GSM4929191,Batch 3 PBMC 10x Lane 7 TCR,GSM4929191,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B3_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873814|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556646,NONE,GSE161918,0 +GSM4929192,Batch 3 PBMC 10x Lane 8 TCR,GSM4929192,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B3_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873813|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556647,NONE,GSE161918,0 +GSM4929193,Batch 3 PBMC 10x Lane 9 TCR,GSM4929193,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B3_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873812|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556648,NONE,GSE161918,0 +GSM4929194,Batch 3 PBMC 10x Lane 10 TCR,GSM4929194,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B3_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873422|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556649,NONE,GSE161918,0 +GSM4929195,Batch 3 PBMC 10x Lane 11 TCR,GSM4929195,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B3_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873421|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556650,NONE,GSE161918,0 +GSM4929196,Batch 3 PBMC 10x Lane 12 TCR,GSM4929196,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B3_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873420|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556651,NONE,GSE161918,0 +GSM4929197,Batch 3 PBMC 10x Lane 13 TCR,GSM4929197,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B3_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873419|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556652,NONE,GSE161918,0 +GSM4929198,Batch 3 PBMC 10x Lane 14 TCR,GSM4929198,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B3_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873418|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556653,NONE,GSE161918,0 +GSM4929199,Batch 3 PBMC 10x Lane 15 TCR,GSM4929199,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B3_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873417|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556654,NONE,GSE161918,0 +GSM4929200,Batch 3 PBMC 10x Lane 16 TCR,GSM4929200,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B3_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873416|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556655,NONE,GSE161918,0 +GSM4929201,Batch 3 PBMC 10x Lane 17 TCR,GSM4929201,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B3_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873415|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556656,NONE,GSE161918,0 +GSM4929202,Batch 3 PBMC 10x Lane 18 TCR,GSM4929202,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B3_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873414|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556657,NONE,GSE161918,0 +GSM4929203,Batch 3 PBMC 10x Lane 19 TCR,GSM4929203,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B3_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873413|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556658,NONE,GSE161918,0 +GSM4929204,Batch 3 PBMC 10x Lane 20 TCR,GSM4929204,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B3_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873412|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556659,NONE,GSE161918,0 +GSM4929205,Batch 3 PBMC 10x Lane 21 TCR,GSM4929205,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B3_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873411|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556660,NONE,GSE161918,0 +GSM4929206,Batch 3 PBMC 10x Lane 22 TCR,GSM4929206,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B3_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873410|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556661,NONE,GSE161918,0 +GSM4929207,Batch 3 PBMC 10x Lane 23 TCR,GSM4929207,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B3_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873409|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556662,NONE,GSE161918,0 +GSM4929208,Batch 3 PBMC 10x Lane 24 TCR,GSM4929208,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,T cell receptor VDJ region reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.|B3_TCR_filtered_contig_annotations.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873408|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556663,NONE,GSE161918,0 +GSM4929209,Batch 3 PBMC 10x Lane 1 RNA,GSM4929209,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873407|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556664,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929209/suppl/GSM4929209_B3_10xlane01_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929210,Batch 3 PBMC 10x Lane 2 RNA,GSM4929210,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873406|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556665,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929210/suppl/GSM4929210_B3_10xlane02_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929211,Batch 3 PBMC 10x Lane 3 RNA,GSM4929211,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873405|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556666,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929211/suppl/GSM4929211_B3_10xlane03_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929212,Batch 3 PBMC 10x Lane 4 RNA,GSM4929212,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873404|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556667,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929212/suppl/GSM4929212_B3_10xlane04_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929213,Batch 3 PBMC 10x Lane 5 RNA,GSM4929213,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873403|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556668,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929213/suppl/GSM4929213_B3_10xlane05_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929214,Batch 3 PBMC 10x Lane 6 RNA,GSM4929214,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873402|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556669,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929214/suppl/GSM4929214_B3_10xlane06_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929215,Batch 3 PBMC 10x Lane 7 RNA,GSM4929215,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873401|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556670,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929215/suppl/GSM4929215_B3_10xlane07_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929216,Batch 3 PBMC 10x Lane 8 RNA,GSM4929216,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873400|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556671,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929216/suppl/GSM4929216_B3_10xlane08_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929217,Batch 3 PBMC 10x Lane 9 RNA,GSM4929217,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873399|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556672,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929217/suppl/GSM4929217_B3_10xlane09_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929218,Batch 3 PBMC 10x Lane 10 RNA,GSM4929218,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873398|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556673,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929218/suppl/GSM4929218_B3_10xlane10_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929219,Batch 3 PBMC 10x Lane 11 RNA,GSM4929219,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873397|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556674,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929219/suppl/GSM4929219_B3_10xlane11_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929220,Batch 3 PBMC 10x Lane 12 RNA,GSM4929220,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873396|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556675,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929220/suppl/GSM4929220_B3_10xlane12_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929221,Batch 3 PBMC 10x Lane 13 RNA,GSM4929221,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873395|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556676,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929221/suppl/GSM4929221_B3_10xlane13_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929222,Batch 3 PBMC 10x Lane 14 RNA,GSM4929222,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873439|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556677,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929222/suppl/GSM4929222_B3_10xlane14_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929223,Batch 3 PBMC 10x Lane 15 RNA,GSM4929223,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873438|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556678,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929223/suppl/GSM4929223_B3_10xlane15_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929224,Batch 3 PBMC 10x Lane 16 RNA,GSM4929224,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873855|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556679,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929224/suppl/GSM4929224_B3_10xlane16_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929225,Batch 3 PBMC 10x Lane 17 RNA,GSM4929225,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873854|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556680,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929225/suppl/GSM4929225_B3_10xlane17_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929226,Batch 3 PBMC 10x Lane 18 RNA,GSM4929226,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873889|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556681,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929226/suppl/GSM4929226_B3_10xlane18_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929227,Batch 3 PBMC 10x Lane 19 RNA,GSM4929227,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873888|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556682,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929227/suppl/GSM4929227_B3_10xlane19_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929228,Batch 3 PBMC 10x Lane 20 RNA,GSM4929228,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873887|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556683,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929228/suppl/GSM4929228_B3_10xlane20_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929229,Batch 3 PBMC 10x Lane 21 RNA,GSM4929229,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873886|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556684,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929229/suppl/GSM4929229_B3_10xlane21_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929230,Batch 3 PBMC 10x Lane 22 RNA,GSM4929230,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873885|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556685,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929230/suppl/GSM4929230_B3_10xlane22_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929231,Batch 3 PBMC 10x Lane 23 RNA,GSM4929231,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873884|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556686,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929231/suppl/GSM4929231_B3_10xlane23_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929232,Batch 3 PBMC 10x Lane 24 RNA,GSM4929232,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Sorted,mixed,mRNA (cDNA) reads from mulitmodal scRNAseq,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","Multple donor samples are combined within each lane; demultiplexing information is in the demuxletResults.tar.gz and allbatches.HTOandTimepointMetadata.csv supplemental files. A filtered dataset of all cells is provided as a Seurat object in R data format, as supplemental file AllBatches_SeuratObj.rds.","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873883|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556687,ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM4929nnn/GSM4929232/suppl/GSM4929232_B3_10xlane24_RNA_filtered_feature_bc_matrix.h5,GSE161918,0 +GSM4929233,Batch 1 bulkRNAseq AA220014,GSM4929233,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Healthy Control,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B1_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873880|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556688,NONE,GSE161918,0 +GSM4929234,Batch1 bulkRNAseq AA220534,GSM4929234,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Healthy Control,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B1_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873879|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556689,NONE,GSE161918,0 +GSM4929235,Batch 1 bulk RNAseq AA220907,GSM4929235,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Healthy Control,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B1_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873878|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556690,NONE,GSE161918,0 +GSM4929236,Batch 1 bulk RNAseq CHI-014,GSM4929236,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Technical Control,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B1_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873877|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556691,NONE,GSE161918,0 +GSM4929237,Batch 1 bulk RNAseq HGR0000071,GSM4929237,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B1_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873876|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556692,NONE,GSE161918,0 +GSM4929238,Batch 1 bulk RNAseq HGR0000074,GSM4929238,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B1_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873875|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556693,NONE,GSE161918,0 +GSM4929239,Batch 1 bulk RNAseq HGR0000078_T0,GSM4929239,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B1_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873882|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556694,NONE,GSE161918,0 +GSM4929240,Batch 1 bulk RNAseq HGR0000078_T1,GSM4929240,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 1,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B1_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873881|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556695,NONE,GSE161918,0 +GSM4929241,Batch 1 bulk RNAseq HGR0000079_T0,GSM4929241,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B1_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873874|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556696,NONE,GSE161918,0 +GSM4929242,Batch 1 bulk RNAseq HGR0000079_T1,GSM4929242,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 1,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B1_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873873|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556697,NONE,GSE161918,0 +GSM4929243,Batch 1 bulk RNAseq HGR0000083_T0,GSM4929243,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B1_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873904|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556698,NONE,GSE161918,0 +GSM4929244,Batch 1 bulk RNAseq HGR0000083_T1,GSM4929244,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 1,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B1_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873903|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556699,NONE,GSE161918,0 +GSM4929245,Batch 1 bulk RNAseq HGR0000089,GSM4929245,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B1_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873902|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556700,NONE,GSE161918,0 +GSM4929246,Batch 1 bulk RNAseq HGR0000091,GSM4929246,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B1_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873901|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556701,NONE,GSE161918,0 +GSM4929247,Batch 1 bulk RNAseq HGR0000093_T0,GSM4929247,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B1_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873900|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556702,NONE,GSE161918,0 +GSM4929248,Batch 1 bulk RNAseq HGR0000093_T1,GSM4929248,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 1,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B1_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873899|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556703,NONE,GSE161918,0 +GSM4929249,Batch 1 bulk RNAseq HGR0000098,GSM4929249,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B1_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873898|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556704,NONE,GSE161918,0 +GSM4929250,Batch 1 bulk RNAseq HGR0000110,GSM4929250,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B1_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873897|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556705,NONE,GSE161918,0 +GSM4929251,Batch 1 bulk RNAseq HGR0000136,GSM4929251,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B1_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873896|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556706,NONE,GSE161918,0 +GSM4929252,Batch 1 bulk RNAseq HGR0000137,GSM4929252,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B1_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873895|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556707,NONE,GSE161918,0 +GSM4929253,Batch 1 bulk RNAseq HGR0000141,GSM4929253,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B1_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873894|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556708,NONE,GSE161918,0 +GSM4929254,Batch 1 bulk RNAseq HGR0000143,GSM4929254,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B1_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873429|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556709,NONE,GSE161918,0 +GSM4929255,Batch 1 bulk RNAseq IM000040,GSM4929255,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Healthy Control,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B1_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873428|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556710,NONE,GSE161918,0 +GSM4929256,Batch 2 bulk RNAseq CHI014_B2,GSM4929256,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Healthy Control,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873427|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556711,NONE,GSE161918,0 +GSM4929257,Batch 2 bulk RNAseq HD_ML_bc,GSM4929257,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Technical test sample,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873426|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556712,NONE,GSE161918,0 +GSM4929258,Batch 2 bulk RNAseq HD_ML_PBMC,GSM4929258,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Healthy Control,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873425|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556713,NONE,GSE161918,0 +GSM4929259,Batch 2 bulk RNAseq HD_VO_bc,GSM4929259,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Technical test sample,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873424|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556714,NONE,GSE161918,0 +GSM4929260,Batch 2 bulk RNAseq HD_VO_PBMC,GSM4929260,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Healthy Control,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873423|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556715,NONE,GSE161918,0 +GSM4929261,Batch 2 bulk RNAseq HGR0000042_T0,GSM4929261,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873456|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556716,NONE,GSE161918,0 +GSM4929262,Batch 2 bulk RNAseq HGR0000042_T1,GSM4929262,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 1,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873455|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556717,NONE,GSE161918,0 +GSM4929263,Batch 2 bulk RNAseq HGR0000069_T0,GSM4929263,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873454|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556718,NONE,GSE161918,0 +GSM4929264,Batch 2 bulk RNAseq HGR0000069_T1,GSM4929264,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 1,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873453|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556719,NONE,GSE161918,0 +GSM4929265,Batch 2 bulk RNAseq HGR0000069_T2,GSM4929265,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 2,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873452|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556720,NONE,GSE161918,0 +GSM4929266,Batch 2 bulk RNAseq HGR0000092_T0,GSM4929266,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873451|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556721,NONE,GSE161918,0 +GSM4929267,Batch 2 bulk RNAseq HGR0000092_T1,GSM4929267,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 1,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873450|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556722,NONE,GSE161918,0 +GSM4929268,Batch 2 bulk RNAseq HGR0000094_T0,GSM4929268,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873449|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556723,NONE,GSE161918,0 +GSM4929269,Batch 2 bulk RNAseq HGR0000094_T1,GSM4929269,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 1,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873448|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556724,NONE,GSE161918,0 +GSM4929270,Batch 2 bulk RNAseq HGR0000115_T1,GSM4929270,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 1,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873447|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556725,NONE,GSE161918,0 +GSM4929271,Batch 2 bulk RNAseq HGR0000124_T0,GSM4929271,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873446|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556726,NONE,GSE161918,0 +GSM4929272,Batch 2 bulk RNAseq HGR0000124_T1,GSM4929272,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 1,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873445|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556727,NONE,GSE161918,0 +GSM4929273,Batch 2 bulk RNAseq HGR0000144_T0,GSM4929273,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873444|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556728,NONE,GSE161918,0 +GSM4929274,Batch 2 bulk RNAseq HGR0000144_T2,GSM4929274,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 2,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873443|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556729,NONE,GSE161918,0 +GSM4929275,Batch 2 bulk RNAseq HGR0000391_T0,GSM4929275,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873437|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556730,NONE,GSE161918,0 +GSM4929276,Batch 2 bulk RNAseq HGR0000395_T0,GSM4929276,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873436|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556731,NONE,GSE161918,0 +GSM4929277,Batch 2 bulk RNAseq HGR0000418_T1,GSM4929277,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 1,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873435|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556732,NONE,GSE161918,0 +GSM4929278,Batch 2 bulk RNAseq SHD2,GSM4929278,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Healthy Control,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873434|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556733,NONE,GSE161918,0 +GSM4929279,Batch 2 bulk RNAseq SHD4,GSM4929279,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Healthy Control,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873433|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556734,NONE,GSE161918,0 +GSM4929280,Batch 2 bulk RNAseq SHD7,GSM4929280,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Healthy Control,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873432|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556735,NONE,GSE161918,0 +GSM4929281,Batch 2 bulk RNAseq SHD8,GSM4929281,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Healthy Control (excluded due to abnormal lymphoma-like B cells found upon analysis),mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873431|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556736,NONE,GSE161918,0 +GSM4929282,Batch 3 bulk RNAseq CHI014_B3,GSM4929282,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Technical Control,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873430|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556737,NONE,GSE161918,0 +GSM4929283,Batch 3 bulk RNAseq HGR0000051_T0,GSM4929283,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873892|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556738,NONE,GSE161918,0 +GSM4929284,Batch 3 bulk RNAseq HGR0000051_T1,GSM4929284,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 1,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873891|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556739,NONE,GSE161918,0 +GSM4929285,Batch 3 bulk RNAseq HGR0000051_T2,GSM4929285,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 2,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873890|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556740,NONE,GSE161918,0 +GSM4929286,Batch 3 bulk RNAseq HGR0000051_T3,GSM4929286,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 3,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873921|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556741,NONE,GSE161918,0 +GSM4929287,Batch 3 bulk RNAseq HGR0000101_T0,GSM4929287,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873920|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556742,NONE,GSE161918,0 +GSM4929288,Batch 3 bulk RNAseq HGR0000101_T1,GSM4929288,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 1,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873919|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556743,NONE,GSE161918,0 +GSM4929289,Batch 3 bulk RNAseq HGR0000101_T3,GSM4929289,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 3,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873918|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556744,NONE,GSE161918,0 +GSM4929290,Batch 3 bulk RNAseq HGR0000102_T0,GSM4929290,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873917|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556745,NONE,GSE161918,0 +GSM4929291,Batch 3 bulk RNAseq HGR0000102_T1,GSM4929291,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 1,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873916|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556746,NONE,GSE161918,0 +GSM4929292,Batch 3 bulk RNAseq HGR0000134_T0,GSM4929292,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873915|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556747,NONE,GSE161918,0 +GSM4929293,Batch 3 bulk RNAseq HGR0000134_T1,GSM4929293,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 1,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873914|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556748,NONE,GSE161918,0 +GSM4929294,Batch 3 bulk RNAseq HGR0000135_T0,GSM4929294,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873913|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556749,NONE,GSE161918,0 +GSM4929295,Batch 3 bulk RNAseq HGR0000135_T1,GSM4929295,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 1,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873912|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556750,NONE,GSE161918,0 +GSM4929296,Batch 3 bulk RNAseq HGR0000142_T0,GSM4929296,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873911|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556751,NONE,GSE161918,0 +GSM4929297,Batch 3 bulk RNAseq HGR0000392_T0,GSM4929297,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 0,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873925|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556752,NONE,GSE161918,0 +GSM4929298,Batch 3 bulk RNAseq HGR0000392_T3,GSM4929298,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 3,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873924|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556753,NONE,GSE161918,0 +GSM4929299,Batch 3 bulk RNAseq HGR0000429_T1,GSM4929299,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 1,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873910|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556754,NONE,GSE161918,0 +GSM4929300,Batch 3 bulk RNAseq HGR0000429_T2,GSM4929300,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 2,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873909|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556755,NONE,GSE161918,0 +GSM4929301,Batch 3 bulk RNAseq HGR0000430_T1,GSM4929301,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Timepoint 1,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873908|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556756,NONE,GSE161918,0 +GSM4929302,Batch 3 bulk RNAseq SHD1,GSM4929302,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Healthy Control,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873907|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556757,NONE,GSE161918,0 +GSM4929303,Batch 3 bulk RNAseq SHD3,GSM4929303,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Healthy Control,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873906|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556758,NONE,GSE161918,0 +GSM4929304,Batch 3 bulk RNAseq SHD5,GSM4929304,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Healthy Control,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873905|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556759,NONE,GSE161918,0 +GSM4929305,Batch 3 bulk RNAseq SHD6,GSM4929305,Public on Feb 10 2021,Nov 23 2020,Sep 05 2023,SRA,1,Peripheral Blood Mononuclear Cells,Homo sapiens,9606,Unsorted,Healthy Control,mRNA,total RNA,"Frozen PBMC samples were thawed, recovered and washed using RPMI media with 10% FBS and 10mg/mL DNase I (STEMCELL 07470). Samples from different donors were pooled evenly before staining; single cells can be demultiplexed in silico using individual specific single nucleotide polymorphism (SNP) information during data analysis (see below; demultiplexing for each lane is provided in the *demux.best supplemental files). Multiple cell pools were prepared, such that cells from the same individual but different timepoints would be in different pools. PBMC pools were Fc blocked (Human TruStain FcX, BioLegend 422302) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS). This round of hashtag staining allows the different pools to be identified in the analysis, which when combined with the SNP-based demultiplexing, allows full identification of each sample. A fraction of the combined cells was used for sorting total B, T, CD14+ monocyte and CD14- innate cell populations (batch 1) or non-naïve T and B cells (batches 2 and 3). Separately for the unsorted and sorted cell fractions, hashtagged PBMC pools were combined and cells were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 192 surface proteins. Then, cells were washed three times, resuspended in PBS, and counted before proceeding immediately to the single cell partition step. Flow cytometry sorting to enrich certain cell populations was performed as follows: Pooled PBMC samples from different donors were washed with PBS and incubated with Zombie Red Fixable viability dye (1:1000 in PBS, BioLgend) for 20 minutes at 4°C protected from light. Then cells were washed with flow staining buffer (10% FBS in PBS) and Fc blocked (Human TruStain FcX, BioLegend) for 15 minutes on ice. For experimental batches 2 and 3, the non-naïve B- and T- cell populations were sorted. The fluorescence-labeled antibody cocktail against human CD45(APC/Cyanine7, BioLegend 368516), CD3(AF488, BioLegend 344810), CD19(APC, BioLegend 363006), CCR7(BV786, BioLegend 353230), CD95(BV650, BioLgend 305642), IgD(PerCP-Cy5.5, BioLegend 348208) and CD27(PE/Cyanine7, BioLegend 356412) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). Non-naïve B cell population was gated by CD45+CD19+IgD- or CD27+ and non-naïve T cell population was gated by CD45+CD3+CCR7low or CD95+. Sorted cells were CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step. For experimental batch 1, total B, T, CD14+ monocyte and CD14- innate cell populations were sorted and pooled with unsorted PBMCs by ratio 1:1:1:1:1 to balance the number of each population captured in the final pool for CITE-seq assay. The fluorescence-labeled antibody cocktail against human CD14(BV421, BioLegend 325628), CD3(AF488, BioLegend 344810), CD45(PE/Cyanine7, BioLegend 393408), CD19(APC, BioLegend 363006) and Totalseq-C human hashtag antibodies(BioLegend) were added at the end of blocking, and incubated for 20 minutes at 4°C in the dark. Cells were washed and sorted on a BD Aria sorter in Biosafety Level 3 (BSL3) lab (BD Biosciences). B cell population was gated by CD45+CD19+CD3-; T cell population was gated by CD45+CD3+CD19-; monocyte population was gated by CD45+CD3-CD19-CD14+; other myeloid cells were gated by CD45+CD3-CD19-CD14-. Sorted 4 populations and unsorted PBMCs were pooled evenly. Then the final pool was CITE-seq stained, washed three times, resuspended in PBS and counted before proceeding to the single cell partition step.|PBMC samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5’ Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry (10x Genomics, Pleasanton, CA). The RT step was conducted in the Veriti Thermo Cycler (ThermoFisher Scientific, Waltham, MA). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA) and quantified using Qubit Fluorometric (ThermoFisher). 10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. Sequencing saturation of the libraries ranged from approximately 60-80% for the cDNA and 20-40% for the surface protein tag libraries.","For each sample, a sample of 100,000 cells was processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD; catalogue number 217084) and standard RNA sequencing libraries were generated using Illumina Truseq library (Illuimna, San Diego, CA; 20020595) preparation kits. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol: Nucleic Acids Research, Volume 47, Issue 21, 02 December 2019, Page e136. These SNP calls were then used along with the demuxlet software (https://github.com/statgen/demuxlet) to demultiplex the single cell multimodal RNAseq data (see supplemental demux.best files). Counts per gene were generated using htseq count (version 0.9.1) and the UCSC hg19 genome annotation.|B2_B3_bulkRNAseq_countmtx.csv","fastq generation: Illumina bcl2fastq 2.20|mapping, CellRanger 3.1.0 (10x Genomics)|sample demultiplexing: popscle demuxlet (https://github.com/statgen/popscle)|secondary analysis: Seurat 3.1.0 (R package)|T cell receptor VDJ analysis: CellRanger 3.1.0|One anonymized donor (SDH8) was excluded from further analysis, as we discovered an extremely expanded B phenotype consistent lymphoma in this sample. An additional leukopack-derived PBMC sample (CHI014, anonymized and without demographic information) was used in each experimental batch as a technical control, but not used in later comparisons. Samples HDVO_bc and HDML_bc in Batch 2 were used as a test of cyropreserving buffy coat samples, and were not used in the furtehr analysis.|Genome_build: hg19 (cellranger version 1.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build#hg19_1.2.0)|Supplementary_files_format_and_content: CellRanger count matrices in h5 data format; T cell clonal analysis, comma seperated values",GPL24676,"Andrew,,Martins",andrew.martins@yale.edu,Immunobiology,Yale School of Medicine,100 College Street Rm 1155,New Haven,Connecticut,06510,USA,Illumina NovaSeq 6000,cDNA,transcriptomic,RNA-Seq,BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN16873923|SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX9556760,NONE,GSE161918,0 diff --git a/paper-releases/pbisc-diffusion/updates/20261002T124820Z-crosscohort-liu-default/diffusion/dataset/server_1/dependency/fb76b84fa119597e/sc_samples.csv b/paper-releases/pbisc-diffusion/updates/20261002T124820Z-crosscohort-liu-default/diffusion/dataset/server_1/dependency/fb76b84fa119597e/sc_samples.csv new file mode 100644 index 0000000000000000000000000000000000000000..e2251b6d6eff7c64b1d59f5c43414858160a9e0c --- /dev/null +++ b/paper-releases/pbisc-diffusion/updates/20261002T124820Z-crosscohort-liu-default/diffusion/dataset/server_1/dependency/fb76b84fa119597e/sc_samples.csv @@ -0,0 +1,71 @@ +sc_sample,Batch,Donor,Subject,Timepoint,Class,Age,Gender,Ward,Status,symptom_type,hospital_days,day_from_symptom_onset_to_hospitalization,number_of_days_hospitalized,computed_ethnicity,ever_admitted_to_icu,severity,days_of_symptoms_onset,condition,outcome,DDimer,CRP,material_type,sample_bsi_id,sample_alt_id,sample_label,batch,days_since_hospitalized,days_since_onset,severity.outcome,PC1,PC1_cat,cond_group,onset_group,intubation_vent,PC1_onset_group,sample_name,Sorted,BatchSort,n_cells_object,n_sorted_dropped,n_unsorted,n_unsorted_hto_singlet,n_unsorted_hto_doublet,n_unsorted_hto_negative,n_unsorted_not_demuxlet_singlet,n_selected,hashtags,kind +B1_AA220014_HC,B1,AA220014,AA220014,HC,HC,67,Male,Healthy_control,na,HC,0,,,,,,0,,,,,,,,,,,,,,,HC,,,NA_NA,AA220014_HC,N,B1UnSort,2665,2212,453,428,25,0,0,428,HTO1,HC +B1_AA220534_HC,B1,AA220534,AA220534,HC,HC,47,Male,Healthy_control,na,HC,0,,,,,,0,,,,,,,,,,,,,,,HC,,,NA_NA,AA220534_HC,N,B1UnSort,2000,1630,370,353,17,0,0,353,HTO2,HC 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+B1_HGR0000079_T0,B1,HGR0000079,HGR0000079,T0,COVID,37,Female,Infectious_Diseases,Alive,phenotype,27,24,27,EUR,FALSE,Critical,30,COVID19-Negative,alive,854,96.2,PBMC,AA500493 0001,BS-EQ-0006:T0:PBMC,BS-EQ-0006,Batch1,6,30,,,,Critical,late,,NA_late,HGR0000079_T0,N,B1UnSort,1405,1282,123,119,4,0,0,119,HTO2,COVID +B1_HGR0000079_T1,B1,HGR0000079,HGR0000079,T1,COVID,37,Female,Infectious_Diseases,Alive,phenotype,27,24,27,EUR,FALSE,Critical,36,COVID19-Negative,alive,854,96.2,PBMC,AA500518 0001,BS-EQ-0006:T1:PBMC,BS-EQ-0006,Batch1,12,36,,,,Critical,late,,NA_late,HGR0000079_T1,N,B1UnSort,955,799,156,145,11,0,0,145,HTO1,COVID +B1_HGR0000083_T0,B1,HGR0000083,HGR0000083,T0,COVID,55,Male,Tropical_Diseases,Alive,phenotype,11,5,11,EUR,FALSE,Moderate,6,COVID19,alive,589,124.9,PBMC,AA500494 0001,BS-EQ-0002:T0:PBMC,BS-EQ-0002,Batch1,1,6,Moderate-Alive,-1.9508583519753,PC1_low,Moderate,early,,PC1_low_early,HGR0000083_T0,N,B1UnSort,2240,2063,177,165,12,0,0,165,HTO2,COVID 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0001,BS-EQ-0004:T0:PBMC,BS-EQ-0004,Batch1,3,20,Moderate-Alive,-1.41813091808461,PC1_low,Moderate,mid,,PC1_low_mid,HGR0000091_T0,N,B1UnSort,1764,1383,381,368,13,0,0,368,HTO1,COVID +B1_HGR0000093_T0,B1,HGR0000093,HGR0000093,T0,COVID,70,Male,Infectious_Diseases,Alive,phenotype,20,10,20,EUR,FALSE,Critical,11,COVID19,alive,327,1.5,PBMC,AA500498 0001,BS-EQ-0008:T0:PBMC,BS-EQ-0008,Batch1,1,11,Critical-Alive,-0.348888393393188,PC1_low,Critical,early,,PC1_low_early,HGR0000093_T0,N,B1UnSort,1773,1288,485,457,28,0,0,457,HTO2,COVID +B1_HGR0000093_T1,B1,HGR0000093,HGR0000093,T1,COVID,70,Male,Infectious_Diseases,Alive,phenotype,20,10,20,EUR,FALSE,Critical,17,COVID19,alive,327,1.5,PBMC,AA500521 0001,BS-EQ-0008:T1:PBMC,BS-EQ-0008,Batch1,7,17,Critical-Alive,-0.348888393393188,PC1_low,Critical,mid,,PC1_low_mid,HGR0000093_T1,N,B1UnSort,981,812,169,154,15,0,0,154,HTO1,COVID 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0001,BS-EQ-0019:T0:PBMC,BS-EQ-0019,Batch1,1,17,Moderate-Alive,-2.91073693427368,PC1_low,Moderate,mid,,PC1_low_mid,HGR0000136_T0,N,B1UnSort,1956,1626,330,320,10,0,0,320,HTO1,COVID +B1_HGR0000137_T0,B1,HGR0000137,HGR0000137,T0,COVID,79,Female,Infectious_Diseases,Alive,phenotype,3,3,3,EUR,FALSE,Severe,4,COVID19,alive,363,36.5,PBMC,AA500508 0001,BS-EQ-0020:T0:PBMC,BS-EQ-0020,Batch1,1,4,Severe-Alive,-1.2015610057303,PC1_low,Severe,early,,PC1_low_early,HGR0000137_T0,N,B1UnSort,1980,1635,345,329,16,0,0,329,HTO1,COVID +B1_HGR0000141_T0,B1,HGR0000141,HGR0000141,T0,COVID,76,Male,Infectious_Diseases,Deceased,phenotype,,14,,EUR,FALSE,Critical,20,COVID19,deceased,0,294.8,PBMC,AA500509 0001,BS-EQ-0022:T0:PBMC,BS-EQ-0022,Batch1,6,20,Critical-Deceased,3.27623326424634,PC1_high,Critical,mid,,PC1_high_mid,HGR0000141_T0,N,B1UnSort,1807,1534,273,258,15,0,0,258,HTO2,COVID +B1_HGR0000143_T0,B1,HGR0000143,HGR0000143,T0,COVID,54,Female,Infectious_Diseases,Alive,phenotype,34,7,34,EAS,TRUE,Critical,8,COVID19,alive,0,2.3,PBMC,AA500511 0001,BS-EQ-0024:T0:PBMC,BS-EQ-0024,Batch1,1,8,Critical-Alive,1.67405631685952,PC1_high,Critical,early,TRUE,PC1_high_early,HGR0000143_T0,N,B1UnSort,1636,1336,300,289,11,0,0,289,HTO2,COVID +B1_IM000040_HC,B1,IM000040,IM000040,HC,HC,37,Male,Healthy_control,na,HC,0,,,,,,0,,,,,,,,,,,,,,,HC,,,NA_NA,IM000040_HC,N,B1UnSort,2352,1958,394,379,15,0,0,379,HTO1,HC +B2_CHI014_HC,B2,CHI014,CHI014,HC,HC,,na,Healthy_control,na,HC,0,,,,,,0,,,,,,,,,,,,,,,HC,,,NA_NA,CHI014_HC,N,B2UnSort,9222,2423,6799,5815,3,981,0,5815,HTO1|HTO2|HTO3,technical_control +B2_HDML_PBMC_HC,B2,HDML,HDML_PBMC,HC,HC,45,Male,Healthy_control,na,HC,0,,,,,,0,,,,,,,,,,,,,,,HC,,,NA_NA,HDML_PBMC_HC,N,B2UnSort,9618,2268,7350,7191,3,156,0,7191,HTO1,technical_control +B2_HDVO_PBMC_HC,B2,HDVO,HDVO_PBMC,HC,HC,30,Male,Healthy_control,na,HC,0,,,,,,0,,,,,,,,,,,,,,,HC,,,NA_NA,HDVO_PBMC_HC,N,B2UnSort,10568,3884,6684,6664,6,14,0,6664,HTO2,technical_control +B2_HGR0000042_T0,B2,HGR0000042,HGR0000042,T0,COVID,53,Male,Infectious_Diseases,Alive,phenotype,15,5,15,EUR,FALSE,Critical,12,COVID19,alive,0,33,PBMC,AA500488 0001,BS-EQ-0015:T0:PBMC,BS-EQ-0015,Batch2,7,12,Critical-Alive,-0.0144850947759859,PC1_high,Critical,early,,PC1_high_early,HGR0000042_T0,N,B2UnSort,4551,1141,3410,2306,5,1099,0,2306,HTO3,COVID +B2_HGR0000042_T1,B2,HGR0000042,HGR0000042,T1,COVID,53,Male,Infectious_Diseases,Alive,phenotype,15,5,15,EUR,FALSE,Critical,18,COVID19,alive,0,33,PBMC,AA500530 0001,BS-EQ-0015:T1:PBMC,BS-EQ-0015,Batch2,13,18,Critical-Alive,-0.0144850947759859,PC1_high,Critical,mid,,PC1_high_mid,HGR0000042_T1,N,B2UnSort,6641,1944,4697,4663,2,32,0,4663,HTO2,COVID +B2_HGR0000069_T0,B2,HGR0000069,HGR0000069,T0,COVID,54,Male,Infectious_Diseases,Alive,phenotype,10,12,10,EUR,FALSE,Severe,13,COVID19,alive,593,0.8,PBMC,AA500514 0001,BS-EQ-0001:T0:PBMC,BS-EQ-0001,Batch2,1,13,Critical-Alive,1.04716869546426,PC1_high,Severe,early,,PC1_high_early,HGR0000069_T0,N,B2UnSort,6909,2351,4558,4524,3,31,0,4524,HTO2,COVID +B2_HGR0000069_T1,B2,HGR0000069,HGR0000069,T1,COVID,54,Male,Infectious_Diseases,Alive,phenotype,10,12,10,EUR,FALSE,Severe,20,COVID19,alive,593,0.8,PBMC,AA500516 0001,BS-EQ-0001:T1:PBMC,BS-EQ-0001,Batch2,8,20,Critical-Alive,1.04716869546426,PC1_high,Severe,mid,,PC1_high_mid,HGR0000069_T1,N,B2UnSort,6981,1534,5447,4670,3,774,0,4670,HTO1,COVID +B2_HGR0000069_T2,B2,HGR0000069,HGR0000069,T2,COVID,54,Male,Infectious_Diseases,Alive,phenotype,10,12,10,EUR,FALSE,Severe,33,COVID19,alive,593,0.8,PBMC,AA500552 0001,BS-EQ-0001:T2:PBMC,BS-EQ-0001,Batch2,21,33,Critical-Alive,1.04716869546426,PC1_high,Severe,late,,PC1_high_late,HGR0000069_T2,N,B2UnSort,5944,1658,4286,2413,7,1866,0,2413,HTO3,COVID +B2_HGR0000092_T0,B2,HGR0000092,HGR0000092,T0,COVID,45,Male,Infectious_Diseases,Alive,phenotype,15,9,15,EUR,FALSE,Critical,11,COVID19,alive,871,179.7,PBMC,AA500497 0001,BS-EQ-0012:T0:PBMC,BS-EQ-0012,Batch2,2,11,Critical-Alive,-0.52272790656358,PC1_low,Critical,early,,PC1_low_early,HGR0000092_T0,N,B2UnSort,7743,2134,5609,5402,9,198,0,5402,HTO1,COVID +B2_HGR0000092_T1,B2,HGR0000092,HGR0000092,T1,COVID,45,Male,Infectious_Diseases,Alive,phenotype,15,9,15,EUR,FALSE,Critical,18,COVID19,alive,871,179.7,PBMC,AA500531 0001,BS-EQ-0012:T1:PBMC,BS-EQ-0012,Batch2,9,18,Critical-Alive,-0.52272790656358,PC1_low,Critical,mid,,PC1_low_mid,HGR0000092_T1,N,B2UnSort,6429,1938,4491,3015,9,1467,0,3015,HTO3,COVID +B2_HGR0000094_T0,B2,HGR0000094,HGR0000094,T0,COVID,61,Male,Infectious_Diseases,Alive,Comorbid,14,5,14,EUR,FALSE,Critical,6,COVID19-Negative,alive,382,0,PBMC,AA500499 0001,BS-EQ-0007:T0:PBMC,BS-EQ-0007,Batch2,1,6,Critical-Alive,0.726261730620933,PC1_high,Critical,early,,PC1_high_early,HGR0000094_T0,N,B2UnSort,4591,1418,3173,3148,2,23,0,3148,HTO2,COVID +B2_HGR0000094_T1,B2,HGR0000094,HGR0000094,T1,COVID,61,Male,Infectious_Diseases,Alive,Comorbid,14,5,14,EUR,FALSE,Critical,13,COVID19-Negative,alive,382,0,PBMC,AA500532 0001,BS-EQ-0007:T1:PBMC,BS-EQ-0007,Batch2,8,13,Critical-Alive,0.726261730620933,PC1_high,Critical,early,,PC1_high_early,HGR0000094_T1,N,B2UnSort,2554,671,1883,967,2,914,0,967,HTO3,COVID +B2_HGR0000115_T1,B2,HGR0000115,HGR0000115,T1,COVID,24,Male,Infectious_Diseases,Alive,phenotype,9,3,9,EUR,FALSE,Severe,11,COVID19,alive,0,14.8,PBMC,AA500535 0001,BS-EQ-0032:T1:PBMC,BS-EQ-0032,Batch2,8,11,Severe-Alive,-2.76779602430212,PC1_low,Severe,early,,PC1_low_early,HGR0000115_T1,N,B2UnSort,8138,2823,5315,5285,1,29,0,5285,HTO2,COVID +B2_HGR0000124_T0,B2,HGR0000124,HGR0000124,T0,COVID,79,Male,Infectious_Diseases,Alive,phenotype,44,8,44,EUR,FALSE,Critical,36,COVID19,alive,0,0,PBMC,AA500504 0001,BS-EQ-0021:T0:PBMC,BS-EQ-0021,Batch2,28,36,,,,Critical,late,,NA_late,HGR0000124_T0,N,B2UnSort,14056,4842,9214,9135,6,73,0,9135,HTO2,COVID +B2_HGR0000124_T1,B2,HGR0000124,HGR0000124,T1,COVID,79,Male,Infectious_Diseases,Alive,phenotype,44,8,44,EUR,FALSE,Critical,49,COVID19,alive,0,0,PBMC,AA500537 0001,BS-EQ-0021:T1:PBMC,BS-EQ-0021,Batch2,41,49,,,,Critical,late,,NA_late,HGR0000124_T1,N,B2UnSort,5969,1928,4041,2617,1,1423,0,2617,HTO3,COVID +B2_HGR0000144_T0,B2,HGR0000144,HGR0000144,T0,COVID,84,Female,Infectious_Diseases,Alive,phenotype,25,12,25,EUR,FALSE,Severe,15,COVID19,alive,872,179,PBMC,AA500512 0001,BS-EQ-0028:T0:PBMC,BS-EQ-0028,Batch2,3,15,Severe-Alive,-0.158291992685593,PC1_high,Severe,early,,PC1_high_early,HGR0000144_T0,N,B2UnSort,5288,999,4289,2283,5,2001,0,2283,HTO3,COVID +B2_HGR0000144_T2,B2,HGR0000144,HGR0000144,T2,COVID,84,Female,Infectious_Diseases,Alive,phenotype,25,12,25,EUR,FALSE,Severe,28,COVID19,alive,872,179,PBMC,AA500559 0001,BS-EQ-0028:T2:PBMC,BS-EQ-0028,Batch2,16,28,Severe-Alive,-0.158291992685593,PC1_high,Severe,late,,PC1_high_late,HGR0000144_T2,N,B2UnSort,8949,1691,7258,6434,8,816,0,6434,HTO1,COVID +B2_HGR0000391_T0,B2,HGR0000391,HGR0000391,T0,COVID,66,Male,Infectious_Diseases,Alive,Comorbid,9,5,9,EUR,FALSE,Critical,6,COVID19,alive,220,2.2,PBMC,AA500523 0001,BS-EQ-0029:T0:PBMC,BS-EQ-0029,Batch2,1,6,Critical-Alive,-1.30858460669219,PC1_low,Critical,early,,PC1_low_early,HGR0000391_T0,N,B2UnSort,9292,2382,6910,6846,5,59,0,6846,HTO1,COVID +B2_HGR0000395_T0,B2,HGR0000395,HGR0000395,T0,COVID,50,Male,Tropical_Diseases,Alive,phenotype,8,12,8,EUR,FALSE,Severe,14,COVID19,alive,229,12.1,PBMC,AA500525 0001,BS-EQ-0034:T0:PBMC,BS-EQ-0034,Batch2,2,14,Severe-Alive,-2.51313825051465,PC1_low,Severe,early,,PC1_low_early,HGR0000395_T0,N,B2UnSort,5628,1557,4071,2871,3,1197,0,2871,HTO3,COVID +B2_HGR0000418_T1,B2,HGR0000418,HGR0000418,T1,COVID,50,Male,Infectious_Diseases,Alive,phenotype,14,21,14,EUR,FALSE,Critical,32,COVID19,alive,0,95.4,PBMC,AA500546 0001,BS-EQ-0036:T1:PBMC,BS-EQ-0036,Batch2,11,32,,,,Critical,late,,NA_late,HGR0000418_T1,N,B2UnSort,8474,1828,6646,6313,5,328,0,6313,HTO1,COVID +B2_SHD2_HC,B2,SHD2,SHD2,HC,HC,53,Male,Healthy_control,na,HC,0,,,,,,0,,,,,,,,,,,,,,,HC,,,NA_NA,SHD2_HC,N,B2UnSort,8603,2885,5718,3538,1,2179,0,3538,HTO3,HC +B2_SHD4_HC,B2,SHD4,SHD4,HC,HC,63,Male,Healthy_control,na,HC,0,,,,,,0,,,,,,,,,,,,,,,HC,,,NA_NA,SHD4_HC,N,B2UnSort,10177,2890,7287,7240,7,40,0,7240,HTO1,HC +B2_SHD7_HC,B2,SHD7,SHD7,HC,HC,77,Male,Healthy_control,na,HC,0,,,,,,0,,,,,,,,,,,,,,,HC,,,NA_NA,SHD7_HC,N,B2UnSort,9502,2989,6513,6488,2,23,0,6488,HTO2,HC +B3_CHI014_HC,B3,CHI014,CHI014,HC,HC,,na,Healthy_control,na,HC,0,,,,,,0,,,,,,,,,,,,,,,HC,,,NA_NA,CHI014_HC,N,B3UnSort,14018,4837,9181,7737,87,1357,0,7737,HTO1|HTO2|HTO3|HTO5,technical_control +B3_HGR0000051_T0,B3,HGR0000051,HGR0000051,T0,COVID,55,Male,Tropical_Diseases,Alive,phenotype,14,7,14,EUR,FALSE,Critical,10,COVID19,alive,618,276.8,PBMC,AA500489 0001,BS-EQ-0014:T0:PBMC,BS-EQ-0014,Batch3,3,10,Critical-Alive,0.0824050089906214,PC1_high,Critical,early,,PC1_high_early,HGR0000051_T0,N,B3UnSort,4378,1153,3225,3158,67,0,0,3158,HTO3,COVID +B3_HGR0000051_T1,B3,HGR0000051,HGR0000051,T1,COVID,55,Male,Tropical_Diseases,Alive,phenotype,14,7,14,EUR,FALSE,Critical,14,COVID19,alive,618,276.8,PBMC,AA500515 0001,BS-EQ-0014:T1:PBMC,BS-EQ-0014,Batch3,7,14,Critical-Alive,0.0824050089906214,PC1_high,Critical,early,,PC1_high_early,HGR0000051_T1,N,B3UnSort,6772,1715,5057,5057,0,0,0,5057,HTO2,COVID +B3_HGR0000051_T2,B3,HGR0000051,HGR0000051,T2,COVID,55,Male,Tropical_Diseases,Alive,phenotype,14,7,14,EUR,FALSE,Critical,17,COVID19,alive,618,276.8,PBMC,AA500551 0001,BS-EQ-0014:T2:PBMC,BS-EQ-0014,Batch3,10,17,Critical-Alive,0.0824050089906214,PC1_high,Critical,mid,,PC1_high_mid,HGR0000051_T2,N,B3UnSort,6060,1773,4287,2179,71,2037,0,2179,HTO5,COVID +B3_HGR0000051_T3,B3,HGR0000051,HGR0000051,T3,COVID,55,Male,Tropical_Diseases,Alive,phenotype,14,7,14,EUR,FALSE,Critical,20,COVID19,alive,618,276.8,PBMC,AA500563 0001,BS-EQ-0014:T3:PBMC,BS-EQ-0014,Batch3,13,20,Critical-Alive,0.0824050089906214,PC1_high,Critical,mid,,PC1_high_mid,HGR0000051_T3,N,B3UnSort,7400,2207,5193,5119,74,0,0,5119,HTO1,COVID +B3_HGR0000101_T0,B3,HGR0000101,HGR0000101,T0,COVID,73,Male,Tropical_Diseases,Alive,phenotype,22,7,22,EUR,FALSE,Critical,8,COVID19,alive,5211,68,PBMC,AA500501 0001,BS-EQ-0011:T0:PBMC,BS-EQ-0011,Batch3,1,8,Critical-Alive,2.38816634675649,PC1_high,Critical,early,,PC1_high_early,HGR0000101_T0,N,B3UnSort,2904,890,2014,977,23,1014,0,977,HTO5,COVID +B3_HGR0000101_T1,B3,HGR0000101,HGR0000101,T1,COVID,73,Male,Tropical_Diseases,Alive,phenotype,22,7,22,EUR,FALSE,Critical,14,COVID19,alive,5211,68,PBMC,AA500522 0001,BS-EQ-0011:T1:PBMC,BS-EQ-0011,Batch3,7,14,Critical-Alive,2.38816634675649,PC1_high,Critical,early,,PC1_high_early,HGR0000101_T1,N,B3UnSort,7872,2801,5071,5010,61,0,0,5010,HTO1,COVID +B3_HGR0000101_T3,B3,HGR0000101,HGR0000101,T3,COVID,73,Male,Tropical_Diseases,Alive,phenotype,22,7,22,EUR,FALSE,Critical,27,COVID19,alive,5211,68,PBMC,AA500564 0001,BS-EQ-0011:T3:PBMC,BS-EQ-0011,Batch3,20,27,Critical-Alive,2.38816634675649,PC1_high,Critical,late,,PC1_high_late,HGR0000101_T3,N,B3UnSort,6075,1999,4076,4001,75,0,0,4001,HTO3,COVID +B3_HGR0000102_T0,B3,HGR0000102,HGR0000102,T0,COVID,58,Male,Infectious_Diseases,Alive,phenotype,24,7,24,EUR,FALSE,Critical,10,COVID19,alive,208,4.9,PBMC,AA500502 0001,BS-EQ-0010:T0:PBMC,BS-EQ-0010,Batch3,3,10,Critical-Alive,-1.57139207217568,PC1_low,Critical,early,,PC1_low_early,HGR0000102_T0,N,B3UnSort,7555,2860,4695,4626,69,0,0,4626,HTO3,COVID +B3_HGR0000102_T1,B3,HGR0000102,HGR0000102,T1,COVID,58,Male,Infectious_Diseases,Alive,phenotype,24,7,24,EUR,FALSE,Critical,17,COVID19,alive,208,4.9,PBMC,AA500533 0001,BS-EQ-0010:T1:PBMC,BS-EQ-0010,Batch3,10,17,Critical-Alive,-1.57139207217568,PC1_low,Critical,mid,,PC1_low_mid,HGR0000102_T1,N,B3UnSort,9292,3508,5784,5690,94,0,0,5690,HTO1,COVID +B3_HGR0000134_T0,B3,HGR0000134,HGR0000134,T0,COVID,53,Male,Tropical_Diseases,Alive,Comorbid,8,14,8,EUR,FALSE,Critical,16,COVID19,alive,0,19.3,PBMC,AA500505 0001,BS-EQ-0017:T0:PBMC,BS-EQ-0017,Batch3,2,16,Critical-Alive,-1.92544770891706,PC1_low,Critical,early,,PC1_low_early,HGR0000134_T0,N,B3UnSort,8210,2121,6089,6089,0,0,0,6089,HTO2,COVID +B3_HGR0000134_T1,B3,HGR0000134,HGR0000134,T1,COVID,53,Male,Tropical_Diseases,Alive,Comorbid,8,14,8,EUR,FALSE,Critical,22,COVID19,alive,0,19.3,PBMC,AA500538 0001,BS-EQ-0017:T1:PBMC,BS-EQ-0017,Batch3,8,22,Critical-Alive,-1.92544770891706,PC1_low,Critical,mid,,PC1_low_mid,HGR0000134_T1,N,B3UnSort,9372,2918,6454,2783,84,3587,0,2783,HTO5,COVID +B3_HGR0000135_T0,B3,HGR0000135,HGR0000135,T0,COVID,59,Male,Infectious_Diseases,Alive,phenotype,14,7,14,EUR,FALSE,Critical,10,COVID19,alive,0,30.7,PBMC,AA500506 0001,BS-EQ-0018:T0:PBMC,BS-EQ-0018,Batch3,3,10,Critical-Alive,0.75305315128394,PC1_high,Critical,early,,PC1_high_early,HGR0000135_T0,N,B3UnSort,8841,2772,6069,6069,0,0,0,6069,HTO2,COVID +B3_HGR0000135_T1,B3,HGR0000135,HGR0000135,T1,COVID,59,Male,Infectious_Diseases,Alive,phenotype,14,7,14,EUR,FALSE,Critical,16,COVID19,alive,0,30.7,PBMC,AA500539 0001,BS-EQ-0018:T1:PBMC,BS-EQ-0018,Batch3,9,16,Critical-Alive,0.75305315128394,PC1_high,Critical,early,,PC1_high_early,HGR0000135_T1,N,B3UnSort,7509,2380,5129,5019,110,0,0,5019,HTO3,COVID +B3_HGR0000142_T0,B3,HGR0000142,HGR0000142,T0,COVID,49,Male,Infectious_Diseases,Alive,Comorbid,9,12,9,EUR,FALSE,Critical,12,COVID19,alive,291,16.1,PBMC,AA500510 0001,BS-EQ-0023:T0:PBMC,BS-EQ-0023,Batch3,0,12,Critical-Alive,-0.617734576041256,PC1_low,Critical,early,,PC1_low_early,HGR0000142_T0,N,B3UnSort,7893,2761,5132,2866,80,2186,0,2866,HTO5,COVID +B3_HGR0000392_T0,B3,HGR0000392,HGR0000392,T0,COVID,52,Male,Infectious_Diseases,Alive,phenotype,14,0,14,UNKNOWN,FALSE,Critical,2,COVID19,alive,0,0,PBMC,AA500524 0001,BS-EQ-0033:T0:PBMC,BS-EQ-0033,Batch3,2,2,Critical-Alive,-1.84274100791149,PC1_low,Critical,early,,PC1_low_early,HGR0000392_T0,N,B3UnSort,8061,2650,5411,5355,56,0,0,5355,HTO1,COVID +B3_HGR0000392_T3,B3,HGR0000392,HGR0000392,T3,COVID,52,Male,Infectious_Diseases,Alive,phenotype,14,0,14,UNKNOWN,FALSE,Critical,13,COVID19,alive,0,0,PBMC,AA500565 0001,BS-EQ-0033:T3:PBMC,BS-EQ-0033,Batch3,13,13,Critical-Alive,-1.84274100791149,PC1_low,Critical,early,,PC1_low_early,HGR0000392_T3,N,B3UnSort,7958,3106,4852,1445,62,3345,0,1445,HTO5,COVID +B3_HGR0000429_T1,B3,HGR0000429,HGR0000429,T1,COVID,54,Male,Infectious_Diseases,Alive,phenotype,13,16,13,,FALSE,Severe,26,COVID19,alive,0,124.8,PBMC,AA500549 0001,BS-EQ-0043:T1:PBMC,BS-EQ-0043,Batch3,10,26,Severe-Alive,-1.18921567127169,PC1_low,Severe,late,,PC1_low_late,HGR0000429_T1,N,B3UnSort,10783,3377,7406,7406,0,0,0,7406,HTO2,COVID +B3_HGR0000429_T2,B3,HGR0000429,HGR0000429,T2,COVID,54,Male,Infectious_Diseases,Alive,phenotype,13,16,13,,FALSE,Severe,28,COVID19,alive,0,124.8,PBMC,AA500562 0001,BS-EQ-0043:T2:PBMC,BS-EQ-0043,Batch3,12,28,Severe-Alive,-1.18921567127169,PC1_low,Severe,late,,PC1_low_late,HGR0000429_T2,N,B3UnSort,8031,2940,5091,5000,91,0,0,5000,HTO3,COVID +B3_HGR0000430_T1,B3,HGR0000430,HGR0000430,T1,COVID,77,Female,Tropical_Diseases,Alive,Comorbid,,7,,,FALSE,Critical,20,COVID19,deceased,0,63.5,PBMC,AA500550 0001,BS-EQ-0044:T1:PBMC,BS-EQ-0044,Batch3,13,20,Critical-Deceased,2.76885345317608,PC1_high,Critical,mid,,PC1_high_mid,HGR0000430_T1,N,B3UnSort,5262,1814,3448,1941,73,1434,0,1941,HTO5,COVID +B3_SHD1_HC,B3,SHD1,SHD1,HC,HC,51,Male,Healthy_control,na,HC,0,,,,,,0,,,,,,,,,,,,,,,HC,,,NA_NA,SHD1_HC,N,B3UnSort,9623,3373,6250,6250,0,0,0,6250,HTO2,HC +B3_SHD3_HC,B3,SHD3,SHD3,HC,HC,58,Male,Healthy_control,na,HC,0,,,,,,0,,,,,,,,,,,,,,,HC,,,NA_NA,SHD3_HC,N,B3UnSort,9440,4087,5353,5254,99,0,0,5254,HTO3,HC +B3_SHD5_HC,B3,SHD5,SHD5,HC,HC,73,Male,Healthy_control,na,HC,0,,,,,,0,,,,,,,,,,,,,,,HC,,,NA_NA,SHD5_HC,N,B3UnSort,11962,5358,6604,6524,80,0,0,6524,HTO1,HC +B3_SHD6_HC,B3,SHD6,SHD6,HC,HC,74,Female,Healthy_control,na,HC,0,,,,,,0,,,,,,,,,,,,,,,HC,,,NA_NA,SHD6_HC,N,B3UnSort,9127,3531,5596,2554,70,2972,0,2554,HTO5,HC