| We transduced primary human astrocytes (fetal cortex, ScienCell) with a lentiviral CRISPR activation system (VP64-dSpCas9-VP64) carrying one of five gRNAs, selected the transduced cells with puromycin and kept them in a neuronal reprogramming culture until harvest, 10 days after transduction. The gRNA is recorded in the `group` column, which holds our groups (`geo_genotype` repeats the same labels): non-targeting is the non-targeting control gRNA, and gRNA-hit, gRNA 113, gRNA 155 and gRNA 194 are four different gRNAs that all target the FOXO4 promoter. There are three cultures per gRNA, 15 samples in all, and every sample was collected on day 10, so time is not a variable in this experiment. | |
| Each sample is one astrocyte culture given a single gRNA, made into its own library and sequenced once, so there are no technical replicates. The astrocytes came from three donors, but the donor of each culture was not recorded, so no samples can be linked by donor. The rep1 to rep3 in the sample names only number the three cultures within each gRNA; nothing records that cultures with the same number share a donor, a transduction or a processing day. We have no record of processing or sequencing batches and know of no other variable that matters; the remaining columns are GEO annotations that are identical for all samples or repeat the gRNA and sample labels. | |
| The data are bulk poly(A) RNA-seq, given as gene-level raw read counts from featureCounts with GENCODE v22 annotation; the features are gene symbols. | |
| Our question is which genes are differentially expressed in astrocytes given gRNA-hit compared with astrocytes given the non-targeting control gRNA. All 15 samples are part of this question: the cultures with gRNA 113, gRNA 155 and gRNA 194 come from the same experiment, even though they are not among the groups being compared. | |