{
  "schema_version": "seqcolyte.spec.v1",
  "spec_id": "drop_seq",
  "assay": "Drop-seq",
  "chemistry_version": "unspecified",
  "platform": "illumina",
  "platform_params": {
    "read_type": "short"
  },
  "source_docs": [
    {
      "doc_id": "Drop-seq_author_protocol_v1.1.pdf",
      "title": "Drop-seq Laboratory Protocol v1.1",
      "url": "https://doi.org/10.17504/protocols.io.mkbc4sn",
      "path": "/Users/seqmachines/playground/protocols-test/protocols/drop_seq/Drop-seq_author_protocol_v1.1.pdf",
      "retrieved_date": null
    },
    {
      "doc_id": "drop-seq_supp.pdf",
      "title": "drop-seq_supp.pdf",
      "url": null,
      "path": "/Users/seqmachines/playground/protocols-test/protocols/drop_seq/drop-seq_supp.pdf",
      "retrieved_date": null
    },
    {
      "doc_id": "drop-seq_paper.pdf",
      "title": "drop-seq_paper.pdf",
      "url": null,
      "path": "/Users/seqmachines/playground/protocols-test/protocols/drop_seq/drop-seq_paper.pdf",
      "retrieved_date": null
    }
  ],
  "oligos": [
    {
      "oligo_id": "oligo_barcoded_bead_seqb",
      "name": "Barcoded Bead SeqB (bead capture oligo)",
      "aliases": [],
      "role": "Bead-tethered capture/RT primer: SMART PCR handle + cell barcode + UMI + oligo-dT (recommended go-forward bead; used in retina replicates 4-7)",
      "kind": "assembled",
      "sequence": "TTTTTTTAAGCAGTGGTATCAACGCAGAGTAC[CELL_BARCODE:12][UMI:8]TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT",
      "direction": "5_to_3",
      "components": [
        {
          "name": "Bead-Linker",
          "sequence": "",
          "role": "18-carbon PEG flexible-chain linker tethering the oligo to the Toyopearl bead (sequence not specified)"
        },
        {
          "name": "T-spacer",
          "sequence": "TTTTTTT",
          "role": "spacer"
        },
        {
          "name": "SMART PCR handle",
          "sequence": "AAGCAGTGGTATCAACGCAGAGTAC",
          "role": "constant PCR/sequencing handle (SeqB variant, ends ...GAGTAC)"
        },
        {
          "name": "Cell barcode",
          "sequence": "[CELL_BARCODE:12]",
          "role": "12-bp split-and-pool cell barcode (JJJJJJJJJJJJ; 4^12 = 16,777,216 sequences)"
        },
        {
          "name": "UMI",
          "sequence": "[UMI:8]",
          "role": "8-bp unique molecular identifier (NNNNNNNN; 4^8 = 65,536)"
        },
        {
          "name": "Poly(dT)",
          "sequence": "TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT",
          "role": "30-nt oligo-dT mRNA capture / RT priming"
        }
      ],
      "provenance": "reagent",
      "derivation": null,
      "sequence_source": "llm_extracted_from_docs",
      "evidence": [
        {
          "source_doc": "protocol_docs",
          "locator": "oligo / final library",
          "method": "claude_llm_extraction"
        }
      ],
      "notes": "Table S6. 5'-Bead-Linker-TTTTTTTAAGCAGTGGTATCAACGCAGAGTAC JJJJJJJJJJJJ NNNNNNNN T(30)-3'. Prime Read 1 with Read1CustomSeqB. >10^8 primers per bead."
    },
    {
      "oligo_id": "oligo_barcoded_bead_seqa",
      "name": "Barcoded Bead SeqA (bead capture oligo)",
      "aliases": [],
      "role": "Bead-tethered capture/RT primer variant with a longer handle (...GAGTACGT); used in the human-mouse experiments and retina replicates 1-3",
      "kind": "assembled",
      "sequence": "TTTTTTTAAGCAGTGGTATCAACGCAGAGTACGT[CELL_BARCODE:12][UMI:8]TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT",
      "direction": "5_to_3",
      "components": [
        {
          "name": "Bead-Linker",
          "sequence": "",
          "role": "18-carbon PEG flexible-chain linker (sequence not specified)"
        },
        {
          "name": "T-spacer",
          "sequence": "TTTTTTT",
          "role": "spacer"
        },
        {
          "name": "SMART PCR handle",
          "sequence": "AAGCAGTGGTATCAACGCAGAGTACGT",
          "role": "constant PCR/sequencing handle (SeqA variant, ends ...GAGTACGT)"
        },
        {
          "name": "Cell barcode",
          "sequence": "[CELL_BARCODE:12]",
          "role": "12-bp split-and-pool cell barcode (JJJJJJJJJJJJ)"
        },
        {
          "name": "UMI",
          "sequence": "[UMI:8]",
          "role": "8-bp UMI (NNNNNNNN)"
        },
        {
          "name": "Poly(dT)",
          "sequence": "TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT",
          "role": "30-nt oligo-dT"
        }
      ],
      "provenance": "reagent",
      "derivation": null,
      "sequence_source": "llm_extracted_from_docs",
      "evidence": [
        {
          "source_doc": "protocol_docs",
          "locator": "oligo / final library",
          "method": "claude_llm_extraction"
        }
      ],
      "notes": "Table S6. 5'-Bead-Linker-TTTTTTTAAGCAGTGGTATCAACGCAGAGTACGT JJJJJJJJJJJJ NNNNNNNN T(30)-3'. Prime Read 1 with Read1CustomSeqA."
    },
    {
      "oligo_id": "oligo_template_switch_oligo",
      "name": "Template_Switch_Oligo (TSO)",
      "aliases": [],
      "role": "Template-switching oligo; its 3\u2032 riboG triplet pairs the untemplated C's added by Maxima H- RT, appending the SMART handle to the cDNA 3\u2032 end",
      "kind": "single",
      "sequence": "AAGCAGTGGTATCAACGCAGAGTGAATGGG",
      "direction": "5_to_3",
      "components": [
        {
          "name": "SMART handle",
          "sequence": "AAGCAGTGGTATCAACGCAGAGT",
          "role": "constant handle"
        },
        {
          "name": "Spacer",
          "sequence": "GAAT",
          "role": "spacer"
        },
        {
          "name": "Riboguanosines",
          "sequence": "GGG",
          "role": "3\u2032 rGrGrG that base-pairs with cDNA 3\u2032 dC overhang"
        }
      ],
      "provenance": "reagent",
      "derivation": null,
      "sequence_source": "llm_extracted_from_docs",
      "evidence": [
        {
          "source_doc": "protocol_docs",
          "locator": "oligo / final library",
          "method": "claude_llm_extraction"
        }
      ],
      "notes": "Table S6: AAGCAGTGGTATCAACGCAGAGTGAAT rGrGrG (folded rGrGrG -> GGG). Used at 2.5 \u00b5M (supp) / 50 \u00b5M stock (protocol) in RT."
    },
    {
      "oligo_id": "oligo_tso_pcr_smart_primer",
      "name": "TSO_PCR / SMART PCR Primer (Template_Switch_PCR primer)",
      "aliases": [],
      "role": "Whole-transcriptome cDNA amplification primer; anneals to the SMART handle present on both cDNA ends after template switching",
      "kind": "single",
      "sequence": "AAGCAGTGGTATCAACGCAGAGT",
      "direction": "5_to_3",
      "components": [
        {
          "name": "SMART handle",
          "sequence": "AAGCAGTGGTATCAACGCAGAGT",
          "role": "PCR primer / handle"
        }
      ],
      "provenance": "reagent",
      "derivation": null,
      "sequence_source": "llm_extracted_from_docs",
      "evidence": [
        {
          "source_doc": "protocol_docs",
          "locator": "oligo / final library",
          "method": "claude_llm_extraction"
        }
      ],
      "notes": "Table S6 TSO_PCR. Called 'SMART PCR PRIMER' (100 \u00b5M) in the protocol; 0.8 \u00b5M in supp."
    },
    {
      "oligo_id": "oligo_p5_tso_hybrid",
      "name": "P5-TSO_Hybrid (New-P5-SMART PCR hybrid oligo)",
      "aliases": [],
      "role": "Library-PCR forward primer after tagmentation; appends the Illumina P5 adapter to the SMART/3\u2032-cDNA end",
      "kind": "assembled",
      "sequence": "AATGATACGGCGACCACCGAGATCTACACGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC",
      "direction": "5_to_3",
      "components": [
        {
          "name": "P5",
          "sequence": "AATGATACGGCGACCACCGAGATCTACAC",
          "role": "Illumina P5 adapter"
        },
        {
          "name": "Bridge",
          "sequence": "GCCTGTCCGCGG",
          "role": "linker/bridge sequence"
        },
        {
          "name": "SMART handle",
          "sequence": "AAGCAGTGGTATCAACGCAGAGTAC",
          "role": "anneals to bead SMART handle; ends with phosphorothioate *A*C"
        }
      ],
      "provenance": "reagent",
      "derivation": null,
      "sequence_source": "llm_extracted_from_docs",
      "evidence": [
        {
          "source_doc": "protocol_docs",
          "locator": "oligo / final library",
          "method": "claude_llm_extraction"
        }
      ],
      "notes": "Table S6 P5-TSO_Hybrid: AATGATACGGCGACCACCGAGATCTACACGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGT*A*C (3\u2032 *A*C = phosphorothioate). Used at 200 nM in Nextera PCR."
    },
    {
      "oligo_id": "oligo_nextera_n701",
      "name": "Nextera_N701 (N70X i7 library primer)",
      "aliases": [],
      "role": "Library-PCR reverse primer after tagmentation; appends the i7 sample index + Illumina P7 adapter to the Nextera (Tn5) end",
      "kind": "assembled",
      "sequence": "CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGG",
      "direction": "5_to_3",
      "components": [
        {
          "name": "P7",
          "sequence": "CAAGCAGAAGACGGCATACGAGAT",
          "role": "Illumina P7 adapter"
        },
        {
          "name": "i7 sample index (N701)",
          "sequence": "TCGCCTTA",
          "role": "8-bp i7 sample index"
        },
        {
          "name": "Nextera Read 2 handle (s7)",
          "sequence": "GTCTCGTGGGCTCGG",
          "role": "anneals to Nextera mosaic-end/s7 region installed by tagmentation"
        }
      ],
      "provenance": "document",
      "derivation": null,
      "sequence_source": "llm_extracted_from_docs",
      "evidence": [
        {
          "source_doc": "protocol_docs",
          "locator": "oligo / final library",
          "method": "claude_llm_extraction"
        }
      ],
      "notes": "Table S6. Condition-1 index in species-contamination experiment. Used at 200 nM in Nextera PCR."
    },
    {
      "oligo_id": "oligo_nextera_n702",
      "name": "Nextera_N702 (i7 library primer)",
      "aliases": [],
      "role": "Alternate i7-indexed library-PCR reverse primer (species-contamination condition 2)",
      "kind": "assembled",
      "sequence": "CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGG",
      "direction": "5_to_3",
      "components": [
        {
          "name": "P7",
          "sequence": "CAAGCAGAAGACGGCATACGAGAT",
          "role": "Illumina P7 adapter"
        },
        {
          "name": "i7 sample index (N702)",
          "sequence": "CTAGTACG",
          "role": "8-bp i7 sample index"
        },
        {
          "name": "Nextera Read 2 handle (s7)",
          "sequence": "GTCTCGTGGGCTCGG",
          "role": "s7 region"
        }
      ],
      "provenance": "document",
      "derivation": null,
      "sequence_source": "llm_extracted_from_docs",
      "evidence": [
        {
          "source_doc": "protocol_docs",
          "locator": "oligo / final library",
          "method": "claude_llm_extraction"
        }
      ],
      "notes": "Table S6."
    },
    {
      "oligo_id": "oligo_nextera_n703",
      "name": "Nextera_N703 (i7 library primer)",
      "aliases": [],
      "role": "Alternate i7-indexed library-PCR reverse primer (species-contamination condition 3)",
      "kind": "assembled",
      "sequence": "CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGG",
      "direction": "5_to_3",
      "components": [
        {
          "name": "P7",
          "sequence": "CAAGCAGAAGACGGCATACGAGAT",
          "role": "Illumina P7 adapter"
        },
        {
          "name": "i7 sample index (N703)",
          "sequence": "TTCTGCCT",
          "role": "8-bp i7 sample index"
        },
        {
          "name": "Nextera Read 2 handle (s7)",
          "sequence": "GTCTCGTGGGCTCGG",
          "role": "s7 region"
        }
      ],
      "provenance": "document",
      "derivation": null,
      "sequence_source": "llm_extracted_from_docs",
      "evidence": [
        {
          "source_doc": "protocol_docs",
          "locator": "oligo / final library",
          "method": "claude_llm_extraction"
        }
      ],
      "notes": "Table S6."
    },
    {
      "oligo_id": "oligo_read1_custom_seq_a",
      "name": "Read1CustomSeqA (custom Read 1 sequencing primer)",
      "aliases": [],
      "role": "Custom Read 1 sequencing primer for libraries made with Barcoded Bead SeqA; primes the cell barcode + UMI read",
      "kind": "single",
      "sequence": "GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTACGT",
      "direction": "5_to_3",
      "components": [
        {
          "name": "Read1CustomSeqA",
          "sequence": "GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTACGT",
          "role": "custom Read 1 primer"
        }
      ],
      "provenance": "document",
      "derivation": null,
      "sequence_source": "llm_extracted_from_docs",
      "evidence": [
        {
          "source_doc": "protocol_docs",
          "locator": "oligo / final library",
          "method": "claude_llm_extraction"
        }
      ],
      "notes": "Table S6. Loaded at 0.3 \u00b5M for Read 1 priming on NextSeq/MiSeq."
    },
    {
      "oligo_id": "oligo_read1_custom_seq_b",
      "name": "Read1CustomSeqB (custom Read 1 sequencing primer)",
      "aliases": [],
      "role": "Custom Read 1 sequencing primer for libraries made with Barcoded Bead SeqB; primes the cell barcode + UMI read",
      "kind": "single",
      "sequence": "GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC",
      "direction": "5_to_3",
      "components": [
        {
          "name": "Read1CustomSeqB",
          "sequence": "GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC",
          "role": "custom Read 1 primer"
        }
      ],
      "provenance": "document",
      "derivation": null,
      "sequence_source": "llm_extracted_from_docs",
      "evidence": [
        {
          "source_doc": "protocol_docs",
          "locator": "oligo / final library",
          "method": "claude_llm_extraction"
        }
      ],
      "notes": "Table S6."
    },
    {
      "oligo_id": "oligo_p7_tso_hybrid",
      "name": "P7-TSO_Hybrid",
      "aliases": [],
      "role": "P7-side hybrid PCR primer used in the bead-primer RT validation (soluble-RNA) experiment to amplify off the SMART handle",
      "kind": "assembled",
      "sequence": "CAAGCAGAAGACGGCATACGAGATCGTGATCGGTCTCGGCGGAAGCAGTGGTATCAACGCAGAGTAC",
      "direction": "5_to_3",
      "components": [
        {
          "name": "P7",
          "sequence": "CAAGCAGAAGACGGCATACGAGAT",
          "role": "Illumina P7 adapter"
        },
        {
          "name": "Index/bridge",
          "sequence": "CGTGATCGGTCTCGGCGG",
          "role": "index + bridge region"
        },
        {
          "name": "SMART handle",
          "sequence": "AAGCAGTGGTATCAACGCAGAGTAC",
          "role": "anneals to SMART handle; 3\u2032 *A*C phosphorothioate"
        }
      ],
      "provenance": "reagent",
      "derivation": null,
      "sequence_source": "llm_extracted_from_docs",
      "evidence": [
        {
          "source_doc": "protocol_docs",
          "locator": "oligo / final library",
          "method": "claude_llm_extraction"
        }
      ],
      "notes": "Table S6: CAAGCAGAAGACGGCATACGAGATCGTGATCGGTCTCGGCGGAAGCAGTGGTATCAACGCAGAGT*A*C. Used at 400 nM with TruSeq_F."
    },
    {
      "oligo_id": "oligo_truseq_f",
      "name": "TruSeq_F (standard Illumina TruSeq primer)",
      "aliases": [],
      "role": "P5/TruSeq Read 1 primer used in the soluble-RNA validation PCR and to prime Read 1 for the synRNA molecular-barcode read",
      "kind": "single",
      "sequence": "AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT",
      "direction": "5_to_3",
      "components": [
        {
          "name": "P5",
          "sequence": "AATGATACGGCGACCACCGAGATCTACAC",
          "role": "Illumina P5 adapter"
        },
        {
          "name": "TruSeq Read 1",
          "sequence": "TCTTTCCCTACACGACGCTCTTCCGATCT",
          "role": "TruSeq Read 1 sequence"
        }
      ],
      "provenance": "document",
      "derivation": null,
      "sequence_source": "llm_extracted_from_docs",
      "evidence": [
        {
          "source_doc": "protocol_docs",
          "locator": "oligo / final library",
          "method": "claude_llm_extraction"
        }
      ],
      "notes": "Table S6: ...CCGATC*T (3\u2032 phosphorothioate T)."
    },
    {
      "oligo_id": "oligo_cust_syn_rna_seq",
      "name": "CustSynRNASeq",
      "aliases": [],
      "role": "Custom Read 2 sequencing primer used in the synRNA bead-barcode-homogeneity experiment to read the 12-bp cell barcode + 8-bp UMI",
      "kind": "single",
      "sequence": "CGGTCTCGGCGGAAGCAGTGGTATCAACGCAGAGTAC",
      "direction": "5_to_3",
      "components": [
        {
          "name": "CustSynRNASeq",
          "sequence": "CGGTCTCGGCGGAAGCAGTGGTATCAACGCAGAGTAC",
          "role": "custom sequencing primer"
        }
      ],
      "provenance": "document",
      "derivation": null,
      "sequence_source": "llm_extracted_from_docs",
      "evidence": [
        {
          "source_doc": "protocol_docs",
          "locator": "oligo / final library",
          "method": "claude_llm_extraction"
        }
      ],
      "notes": "Table S6."
    },
    {
      "oligo_id": "oligo_umi_smart_dt",
      "name": "UMI_SMARTdT",
      "aliases": [],
      "role": "In-solution RT primer (SMART handle + 9-nt UMI + oligo-dT) for the standard in-solution template-switch amplification comparison",
      "kind": "assembled",
      "sequence": "AAGCAGTGGTATCAACGCAGAGTAC[UMI:9]TTTTTTTTTTTTTTTTTTTTTTTT",
      "direction": "5_to_3",
      "components": [
        {
          "name": "SMART handle",
          "sequence": "AAGCAGTGGTATCAACGCAGAGTAC",
          "role": "constant handle"
        },
        {
          "name": "UMI",
          "sequence": "[UMI:9]",
          "role": "9-nt UMI (NNNNNNNNN)"
        },
        {
          "name": "Poly(dT)",
          "sequence": "TTTTTTTTTTTTTTTTTTTTTTTT",
          "role": "24-nt oligo-dT RT priming"
        }
      ],
      "provenance": "document",
      "derivation": null,
      "sequence_source": "llm_extracted_from_docs",
      "evidence": [
        {
          "source_doc": "protocol_docs",
          "locator": "oligo / final library",
          "method": "claude_llm_extraction"
        }
      ],
      "notes": "Table S6: AAGCAGTGGTATCAACGCAGAGTAC NNNNNNNNN T(24)."
    },
    {
      "oligo_id": "oligo_synrna",
      "name": "synRNA (polyadenylated synthetic RNA spike-in)",
      "aliases": [],
      "role": "Polyadenylated synthetic RNA carrying a 20-nt molecular barcode; used to quantify bead primer sites and validate barcode readout",
      "kind": "assembled",
      "sequence": "CCUACACGACGCUCUUCCGAUCU[UMI:20]BAAAAAAAAAAAAAAAAAAAAAAAA",
      "direction": "5_to_3",
      "components": [
        {
          "name": "TruSeq-derived RNA handle",
          "sequence": "CCUACACGACGCUCUUCCGAUCU",
          "role": "RNA sequence homologous to TruSeq Read 1 region (ribo)"
        },
        {
          "name": "Molecular barcode",
          "sequence": "[UMI:20]",
          "role": "20-nt degenerate molecular barcode (rN x20)"
        },
        {
          "name": "Anchor + poly(A)",
          "sequence": "BAAAAAAAAAAAAAAAAAAAAAAAA",
          "role": "rB anchor + poly(A) tail (24 A)"
        }
      ],
      "provenance": "document",
      "derivation": null,
      "sequence_source": "llm_extracted_from_docs",
      "evidence": [
        {
          "source_doc": "protocol_docs",
          "locator": "oligo / final library",
          "method": "claude_llm_extraction"
        }
      ],
      "notes": "Table S6, given as ribonucleotides (folded rX -> X; RNA U retained). Read 1 (TruSeq primer) reads the 20-bp molecular barcode."
    }
  ],
  "final_library": {
    "source_label": "Drop-seq final Illumina library (Barcoded Bead SeqB chemistry; P5-TSO_Hybrid + Nextera N701)",
    "annotated_library_sequence": "AATGATACGGCGACCACCGAGATCTACACGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC[CELL_BARCODE:12][UMI:8]TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT[CDNA]CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[SAMPLE_INDEX:8]ATCTCGTATGCCGTCTTCTGCTTG",
    "library_sequence": "AATGATACGGCGACCACCGAGATCTACACGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC[CELL_BARCODE:12][UMI:8]TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT[CDNA]CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[SAMPLE_INDEX:8]ATCTCGTATGCCGTCTTCTGCTTG",
    "strands": [
      {
        "direction": "5_to_3",
        "source_html": "AATGATACGGCGACCACCGAGATCTACACGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC[CELL_BARCODE:12][UMI:8]TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT[CDNA]CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[SAMPLE_INDEX:8]ATCTCGTATGCCGTCTTCTGCTTG",
        "source_sequence": "AATGATACGGCGACCACCGAGATCTACACGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC[CELL_BARCODE:12][UMI:8]TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT[CDNA]CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[SAMPLE_INDEX:8]ATCTCGTATGCCGTCTTCTGCTTG"
      },
      {
        "direction": "3_to_5",
        "source_html": "TTACTATGCCGCTGGTGGCTCTAGATGTGCGGACAGGCGCCTTCGTCACCATAGTTGCGTCTCATG[CELL_BARCODE:12]'[UMI:8]'AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA[CDNA]'GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG[SAMPLE_INDEX:8]'TAGAGCATACGGCAGAAGACGAAC",
        "source_sequence": "TTACTATGCCGCTGGTGGCTCTAGATGTGCGGACAGGCGCCTTCGTCACCATAGTTGCGTCTCATG[CELL_BARCODE:12]'[UMI:8]'AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA[CDNA]'GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG[SAMPLE_INDEX:8]'TAGAGCATACGGCAGAAGACGAAC"
      }
    ],
    "annotation_lines": [
      "AATGATACGGCGACCACCGAGATCTACAC = P5",
      "GCCTGTCCGCGG = SMART/P5 hybrid bridge",
      "AAGCAGTGGTATCAACGCAGAGTAC = SMART PCR handle (Read 1 primer region)",
      "[CELL_BARCODE:12] = 12-bp cell barcode",
      "[UMI:8] = 8-bp UMI",
      "TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT = poly(dT) (30 nt)",
      "[CDNA] = cDNA insert",
      "CTGTCTCTTATACACATCT = Nextera Read 2 mosaic end (revcomp of ME)",
      "CCGAGCCCACGAGAC = Nextera Read 2 handle (s7)",
      "[SAMPLE_INDEX:8] = i7 sample index",
      "ATCTCGTATGCCGTCTTCTGCTTG = reverse complement of P7"
    ],
    "evidence": [
      {
        "source_doc": "protocol_docs",
        "locator": "Drop-seq final Illumina library (Barcoded Bead SeqB chemistry; P5-TSO_Hybrid + Nextera N701)",
        "method": "claude_llm_extraction"
      }
    ]
  },
  "read_structure": {
    "reads": [
      {
        "read": "R1",
        "primer": "Read1CustomSeqB / Read1CustomSeqA (custom)",
        "template": "bottom",
        "cycles": 20,
        "segments": [
          {
            "name": "Cell barcode",
            "type": "barcode",
            "order": 0,
            "scored": true,
            "provenance": null,
            "whitelist_ref": null,
            "constant_ref": null,
            "notes": null,
            "length": 12
          },
          {
            "name": "UMI",
            "type": "umi",
            "order": 1,
            "scored": true,
            "provenance": null,
            "whitelist_ref": null,
            "constant_ref": null,
            "notes": null,
            "length": 8
          }
        ]
      },
      {
        "read": "I1",
        "primer": "Illumina i7 index primer",
        "template": "top",
        "cycles": 8,
        "segments": [
          {
            "name": "i7 sample index",
            "type": "index",
            "order": 0,
            "scored": false,
            "provenance": null,
            "whitelist_ref": null,
            "constant_ref": null,
            "notes": null,
            "length": 8
          }
        ]
      },
      {
        "read": "R2",
        "primer": "Nextera Read 2 primer",
        "template": "top",
        "cycles": 50,
        "segments": [
          {
            "name": "cDNA insert",
            "type": "insert",
            "order": 0,
            "scored": true,
            "provenance": null,
            "whitelist_ref": null,
            "constant_ref": null,
            "notes": null
          }
        ]
      }
    ]
  },
  "library_generation": [
    {
      "step": 1,
      "title": "mRNA capture on barcoded bead (droplet co-encapsulation + lysis)",
      "summary": "A lysed cell's polyadenylated mRNA hybridizes by its poly(A) tail to the 30-nt oligo-dT of a co-encapsulated barcoded bead.",
      "note": "The co-flow device pairs one cell with one bead in a ~1 nL droplet; lysis buffer (Ficoll/Sarkosyl/Tris/EDTA/DTT) releases mRNA that anneals to the bead-tethered oligo-dT.",
      "product": "|--5'- ...AAGCAGTGGTATCAACGCAGAGTAC[CELL_BARCODE:12][UMI:8]TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT----->\n                                                       3'- AAAAA......[mRNA] -5'"
    },
    {
      "step": 2,
      "title": "Reverse transcription with template switching (STAMP formation)",
      "summary": "Maxima H- RT extends the bead primer into first-strand cDNA and adds untemplated dC's at the 3\u2032 end.",
      "note": "Reverse transcription copies the mRNA into cDNA covalently attached to the bead (a STAMP); the terminal-transferase activity of Maxima H- appends 2-3 dC's to the cDNA 3\u2032 end.",
      "product": "|--5'- ...AAGCAGTGGTATCAACGCAGAGTAC[CELL_BARCODE:12][UMI:8](T)30[CDNA]CCC----->\n                                                       3'- ...........[mRNA template] -5'"
    },
    {
      "step": 3,
      "title": "Template switch appends SMART handle to cDNA 3\u2032 end",
      "summary": "The TSO rGrGrG anneals to the cDNA dC overhang and is copied, placing a SMART handle on the cDNA 3\u2032 end.",
      "note": "Both ends of the first-strand cDNA now carry the AAGCAGTGGTATCAACGCAGAGT SMART handle, enabling single-primer whole-transcriptome PCR.",
      "product": "|--5'- ...AAGCAGTGGTATCAACGCAGAGTAC[CELL_BARCODE:12][UMI:8](T)30[CDNA]CCCATTCACTCTGCGTTGATACCACTGCTT -3'\n                                                                      GGGTAAG......TGACGAA -5' (from TSO)"
    },
    {
      "step": 4,
      "title": "Exonuclease I treatment",
      "summary": "Exonuclease I chews back bead primers that never captured an mRNA, leaving only extended STAMPs.",
      "note": "Single-stranded, unextended oligos are degraded so they cannot participate in PCR; barcode-bearing extended primers (double-stranded at the 5\u2032 handle after RT) are protected.",
      "product": "|--5'- ...AAGCAGTGGTATCAACGCAGAGTAC[CELL_BARCODE:12][UMI:8](T)30[CDNA]CCCATTCACTCTGCGTTGATACCACTGCTT -3'"
    },
    {
      "step": 5,
      "title": "Whole-transcriptome PCR (SMART/TSO_PCR primer)",
      "summary": "A single SMART PCR primer amplifies full-length cDNA off both handle-bearing ends.",
      "note": "Kapa HiFi + TSO_PCR/SMART primer (AAGCAGTGGTATCAACGCAGAGT) produce double-stranded, amplified cDNA averaging 1300-2000 bp.",
      "product": "5'- AAGCAGTGGTATCAACGCAGAGTAC[CELL_BARCODE:12][UMI:8](T)30[CDNA]CCCATTCACTCTGCGTTGATACCACTGCTT -3'\n3'- TTCGTCACCATAGTTGCGTCTCATG[CB']........[UMI'](A)30[CDNA']GGGTAAGTGAGACGCAACTATGGTGACGAA -5'"
    },
    {
      "step": 6,
      "title": "Nextera XT tagmentation (Tn5)",
      "summary": "Tn5 fragments the amplified cDNA and inserts the Nextera mosaic-end adapter.",
      "note": "Tagmentation adds the Nextera Read 2 / s7 handle to internal 3\u2032-biased fragments; only fragments still bearing the 5\u2032 SMART handle can later receive P5, restricting the library to 3\u2032 ends.",
      "product": "5'- AAGCAGTGGTATCAACGCAGAGTAC[CELL_BARCODE:12][UMI:8](T)30[CDNA]CTGTCTCTTATACACATCT -3'\n                                                          <-- Nextera mosaic end (ME) -->"
    },
    {
      "step": 7,
      "title": "Nextera library PCR (P5-TSO_Hybrid + Nextera N70X)",
      "summary": "P5-TSO_Hybrid adds P5 at the SMART end and Nextera N70X adds the i7 index + P7 at the tagmented end.",
      "note": "Because P5 is added only via the SMART handle, exclusively 3\u2032-end cDNA fragments are amplified into the final sequenceable library (~450-680 bp).",
      "product": "5'- AATGATACGGCGACCACCGAGATCTACACGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC[CELL_BARCODE:12][UMI:8]TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT[CDNA]CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[SAMPLE_INDEX:8]ATCTCGTATGCCGTCTTCTGCTTG -3'"
    }
  ],
  "library_sequencing": [
    {
      "read": "Read 1 (cell barcode + UMI)",
      "primer": "Read1CustomSeqB (GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC) \u2014 Read1CustomSeqA for SeqA beads",
      "template": "bottom",
      "cycles": 20,
      "note": "Custom Read 1 primer anneals to the bottom strand across the bridge + SMART handle; its 3' end sits at the barcode boundary and extension reads the 12-bp cell barcode (bases 1-12) then the 8-bp UMI (bases 13-20).",
      "diagram": "   5'-GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC----------------------->\n5'-...GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTACNNNNNNNNNNNNNNNNNNNNTTTTTTTT...[cDNA]-3'\n3'-...CGGACAGGCGCCTTCGTCACCATAGTTGCGTCTCATGNNNNNNNNNNNNNNNNNNNNAAAAAAAA...[cDNA]-5'\n                                           \\___cell BC (12)___/\\_UMI(8)_/"
    },
    {
      "read": "Index 1 (i7 sample index)",
      "primer": "Nextera i7 index sequencing primer (anneals to the s7 handle: 5'-...CCGAGCCCACGAGAC-3'); only when multiplexing",
      "template": "bottom",
      "cycles": 8,
      "note": "The index primer matches the top-strand s7 handle, so it anneals to the bottom strand with its 3' end at the index boundary and reads the 8-bp i7 sample index installed by the Nextera N70X primer.",
      "diagram": "    5'-CCGAGCCCACGAGAC-------->\n5'- ...CCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'\n3'- ...GGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'\n                      reads 8-bp i7 index -->"
    },
    {
      "read": "Read 2 (cDNA)",
      "primer": "Nextera Read 2 / s7 primer (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG)",
      "template": "top",
      "cycles": 50,
      "note": "The s7 Read 2 primer anneals to the top strand across the Nextera mosaic-end + s7 region; its 3' end points into the insert and extension reads back through the 3' cDNA fragment (50 bp; 60 bp in the retina experiment).",
      "diagram": "                  3'-GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG-5'\n5'- ...(dT)30-[cDNA]-CTGTCTCTTATACACATCTCCGAGCCCACGAGAC-[i7]...-3'\n3'- ...(dA)30-[cDNA]-GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG-[i7]...-5'\n        <-----------[reads cDNA insert]"
    }
  ],
  "whitelists": {},
  "build": {
    "builder_version": "llm-generic-1.0",
    "deterministic": false,
    "source_html_sha256": null,
    "extraction_method": "claude_llm_generic",
    "model": "claude-opus-4-8"
  },
  "title": "Drop-seq",
  "description": "Drop-seq is a droplet-based, massively parallel 3\u2032 single-cell RNA-seq method. Individual cells are co-encapsulated with a DNA-barcoded microparticle (\"bead\") in a nanoliter (~1 nL, 125 \u00b5m) aqueous droplet formed by a co-flow microfluidic device; the cell lyses inside the droplet and its polyadenylated mRNAs hybridize to the bead's oligo-dT primers. Each bead carries a common SMART/PCR handle, a 12-bp split-pool cell barcode (one of 4^12 = 16,777,216), an 8-bp UMI, and a 30-bp oligo-dT. After bulk reverse transcription with template switching (STAMP formation), exonuclease treatment, whole-transcriptome PCR, and Nextera XT tagmentation of the 3\u2032 ends, the library is sequenced on Illumina: Read 1 (20 bp) yields the cell barcode + UMI, and Read 2 the cDNA.",
  "reference": {
    "kind": "paper",
    "label": "Drop-seq Laboratory Protocol v1.1",
    "path": null,
    "url": "https://www.protocols.io/view/drop-seq-laboratory-protocol-mkbc4sn",
    "doi": "10.17504/protocols.io.mkbc4sn"
  },
  "publication": {
    "year": 2015,
    "original_publication": {
      "title": "Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets",
      "journal": "Cell",
      "doi": "10.1016/j.cell.2015.05.002",
      "url": "http://dx.doi.org/10.1016/j.cell.2015.05.002"
    },
    "authors": [
      {
        "name": "Evan Z. Macosko",
        "corresponding": true,
        "email": "emacosko@genetics.med.harvard.edu",
        "affiliation": "Department of Genetics, Harvard Medical School; Stanley Center for Psychiatric Research and Program in Medical and Population Genetics, Broad Institute of Harvard and MIT"
      },
      {
        "name": "Anindita Basu",
        "affiliation": "Klarman Cell Observatory, Broad Institute; School of Engineering and Applied Sciences, Harvard University"
      },
      {
        "name": "Rahul Satija",
        "affiliation": "Klarman Cell Observatory, Broad Institute; New York Genome Center; Department of Biology, New York University"
      },
      {
        "name": "James Nemesh",
        "affiliation": "Department of Genetics, Harvard Medical School; Broad Institute"
      },
      {
        "name": "Karthik Shekhar",
        "affiliation": "Klarman Cell Observatory, Broad Institute"
      },
      {
        "name": "Melissa Goldman",
        "affiliation": "Department of Genetics, Harvard Medical School; Stanley Center for Psychiatric Research, Broad Institute"
      },
      {
        "name": "Itay Tirosh",
        "affiliation": "Klarman Cell Observatory, Broad Institute"
      },
      {
        "name": "Allison R. Bialas",
        "affiliation": "The Program in Cellular and Molecular Medicine, Children's Hospital Boston"
      },
      {
        "name": "Nolan Kamitaki",
        "affiliation": "Department of Genetics, Harvard Medical School; Broad Institute"
      },
      {
        "name": "Emily M. Martersteck",
        "affiliation": "Department of Molecular and Cellular Biology and Center for Brain Science, Harvard University"
      },
      {
        "name": "John J. Trombetta",
        "affiliation": "Klarman Cell Observatory, Broad Institute"
      },
      {
        "name": "David A. Weitz",
        "affiliation": "School of Engineering and Applied Sciences and Department of Physics, Harvard University"
      },
      {
        "name": "Joshua R. Sanes",
        "affiliation": "Department of Molecular and Cellular Biology and Center for Brain Science, Harvard University"
      },
      {
        "name": "Alex K. Shalek",
        "affiliation": "Klarman Cell Observatory, Broad Institute; Ragon Institute of MGH, MIT, and Harvard; Institute for Medical Engineering and Science and Department of Chemistry, MIT"
      },
      {
        "name": "Aviv Regev",
        "affiliation": "Klarman Cell Observatory and Department of Biology, MIT; Howard Hughes Medical Institute"
      },
      {
        "name": "Steven A. McCarroll",
        "corresponding": true,
        "email": "mccarroll@genetics.med.harvard.edu",
        "affiliation": "Department of Genetics, Harvard Medical School; Stanley Center for Psychiatric Research and Program in Medical and Population Genetics, Broad Institute of Harvard and MIT"
      }
    ],
    "throughput": {
      "summary": "~10,000 single-cell libraries prepared per hour at 100 cells/\u00b5L (12 hr for 10,000 cells); ~1,200/hr at 12.5 cells/\u00b5L. ~6.5 cents per cell. 44,808 mouse retinal cells profiled into 39 clusters.",
      "cells": "10,000 single-cell libraries in ~12 hr; 44,808 retinal cells analyzed (49,300 STAMPs sequenced)",
      "rna": "At saturating depth ~44,295 transcripts / 6,722 genes per HEK cell and ~26,044 transcripts / 5,663 genes per 3T3 cell; ~12.8% transcript capture efficiency (ERCC); ~10.7% by ddPCR",
      "dna": "Amplified cDNA ~1300-2000 bp; final tagmented library ~450-680 bp; yield 10-30 nM (HEK/3T3)"
    },
    "statistical_model": "Digital transcript counting via UMIs with edit-distance-1 (substitution-only recommended) collapse; single-cell purity and doublet rates estimated from species-mixing (binomial co-encapsulation) experiments.",
    "other": [
      {
        "label": "Primers per bead",
        "value": ">10^8 barcoded primer sites per microparticle"
      },
      {
        "label": "Droplet size",
        "value": "~125 \u00b5m diameter, ~1 nL, generated on a PDMS co-flow device (>100,000 droplets/min)"
      },
      {
        "label": "Bead synthesis",
        "value": "Reverse-direction (5'->3') phosphoramidite synthesis on Toyopearl HW-65S resin by Chemgenes"
      },
      {
        "label": "Capture efficiency",
        "value": "~12.8% (ERCC spike-in), corroborated at ~10.7% by droplet digital PCR"
      },
      {
        "label": "Accession",
        "value": "GEO: GSE63473"
      },
      {
        "label": "Protocol source",
        "value": "Drop-seq Laboratory Protocol v1.1 (Aug 6, 2015), Macosko & Goldman, McCarroll Lab (www.mccarrolllab.com/dropseq)"
      }
    ]
  },
  "modality": "RNA",
  "method_type": "droplet",
  "data_processing": {
    "summary": "Read 1 supplies the 12-bp cell barcode + 8-bp UMI, which are tagged onto the paired cDNA read; the cDNA (Read 2) is aligned to the reference genome, reads are grouped by cell barcode, and UMIs are collapsed per gene to give digital transcript counts \u2014 producing a cell-by-gene digital gene-expression (DGE) matrix that is then clustered. The paper and protocol point to the McCarroll-lab 'Drop-seq tools' software (Nemesh, with help from Wysoker) and an accompanying downloadable informatics guide.",
    "stages": [
      {
        "id": "prep",
        "label": "Barcode Tagging & Trimming"
      },
      {
        "id": "align",
        "label": "Alignment"
      },
      {
        "id": "quant",
        "label": "Quantification"
      },
      {
        "id": "analysis",
        "label": "Analysis"
      }
    ],
    "nodes": [
      {
        "id": "tag_bc",
        "label": "Tag reads with barcode + UMI",
        "tool": "Drop-seq tools",
        "stage": "prep",
        "scope": "bulk",
        "terminal": false,
        "viz_only": false
      },
      {
        "id": "trim_adapter",
        "label": "Trim SMART adapter",
        "tool": "Drop-seq tools",
        "stage": "prep",
        "scope": "bulk",
        "terminal": false,
        "viz_only": false
      },
      {
        "id": "trim_polya",
        "label": "Trim poly(A) tail",
        "tool": "Drop-seq tools",
        "stage": "prep",
        "scope": "bulk",
        "terminal": false,
        "viz_only": false
      },
      {
        "id": "align",
        "label": "Align cDNA to genome",
        "tool": "STAR",
        "stage": "align",
        "scope": "bulk",
        "terminal": false,
        "viz_only": false
      },
      {
        "id": "group_bc",
        "label": "Group reads by cell barcode",
        "tool": "Drop-seq tools",
        "stage": "quant",
        "scope": "bulk",
        "terminal": false,
        "viz_only": false
      },
      {
        "id": "collapse_umi",
        "label": "Collapse UMIs (edit distance 1)",
        "tool": "Drop-seq tools",
        "stage": "quant",
        "scope": "bulk",
        "terminal": false,
        "viz_only": false
      },
      {
        "id": "build_dge",
        "label": "Build DGE matrix",
        "tool": "Drop-seq tools",
        "stage": "quant",
        "scope": "bulk",
        "terminal": false,
        "viz_only": false
      },
      {
        "id": "cluster",
        "label": "Cluster cells",
        "tool": "",
        "stage": "analysis",
        "scope": "bulk",
        "terminal": true,
        "viz_only": false
      },
      {
        "id": "species_mix",
        "label": "Estimate doublet rate",
        "tool": "",
        "stage": "analysis",
        "scope": "bulk",
        "terminal": true,
        "viz_only": false
      }
    ],
    "edges": [
      {
        "from": "tag_bc",
        "to": "trim_adapter",
        "kind": "sequential"
      },
      {
        "from": "trim_adapter",
        "to": "trim_polya",
        "kind": "sequential"
      },
      {
        "from": "trim_polya",
        "to": "align",
        "kind": "sequential"
      },
      {
        "from": "align",
        "to": "group_bc",
        "kind": "sequential"
      },
      {
        "from": "group_bc",
        "to": "collapse_umi",
        "kind": "sequential"
      },
      {
        "from": "collapse_umi",
        "to": "build_dge",
        "kind": "sequential"
      },
      {
        "from": "build_dge",
        "to": "cluster",
        "kind": "branch"
      },
      {
        "from": "build_dge",
        "to": "species_mix",
        "kind": "branch"
      }
    ],
    "statistical_model": "Digital transcript counting via UMIs collapsed at edit distance 1 (substitution-only); single-cell purity and doublet rate estimated from species-mixing (binomial co-encapsulation) analysis."
  }
}