single-cell RNA seq CRISPR capture of neuron VPH gRNAs
iPSC-derived neurons were harvested and prepared into single-cell suspensions following a published protocol50. Cells were split into two batches, with one batch going through a fluorescence-activated cell sorting (FACS) step to sort on the top 40% of green fluorescent protein (GFP) expression to enrich for neurons with greater numbers of gRNAs integrated, and the second batch going directly into the 10x Genomics single-cell library preparation protocol. Sorting on the top 40% of GFP expression resulted in a two-fold increase in the mean number of gRNAs integrated in those cells as compared to unsorted cells. Four lanes were used for the single-cell transcriptome profiling, with two lanes containing GFP-positive sorted cells, and two lanes containing unsorted cells. Roughly 13,000 cells were captured per lane of a 10x Chromium high-throughput chip (Next GEM Chip M) using Chromium Next GEM Single Cell 3' HT Reagent Kits v3.1 (Dual Index) with Feature Barcode technology for CRISPR Screening (10x Genomics, Inc, Document number CG000418, Rev C).