single cell CRISPR analysis in human pluripotent stem cells

Single-cell CRISPR experiment was performed that focused on the pluripotency network in human iPSCs. We selected 23 genes (TFs) that are thought to be involved in pluripotency in hiPSCs and constructed 2 gRNAs per gene. To capture the time-course expression changes, samples were taken from days 2, 3, 4, and 5 after transduction and scRNA-seq analysis was performed. Approximately 5,000 single cells should be captured in each sample to obtain approximately 100 cells per gRNA in the scRNA-seq. Human iPSCs (OILG-Cas9) were transduced with the pooled lentivirus for both gRNA and BFP expression at 8-9% transduction efficiency and maintained until harvesting without passaging. On days 2, 3, 4, and 5 after transduction, 8 × 104 BFP+ cells were collected using an MA900 cell sorter (Sony), then resuspended at 1 × 106 cells/mL in 0.05% BSA in PBS. These cells were then subjected to 5 ’single-cell RNA-seq library preparation using a Chromium Next GEM Single Cell 5’Library & Gel Bead Kit following the manufacturers’ protocol with minor modifications to simultaneously capture guide RNA molecules.