CRISPR_knockout_gDNA_validation
Targeted gDNA amplified by PCR from wild-type and knockout cells. For each knockout, one million cells from the FACS-isolated “Knockout” and “Control(-)” cell populations (Figure S3A) were pelleted by centrifugation and their genomic DNA was phenol-extracted and used as a template to amplify sgRNA-targeted loci using two-step nested PCR (96°C x 3 min; 35 cycles of 96°C x 30 s, 63°C x 30 s, 72°C x 1 min; 72°C x 10 min; 4°C) using the primer pairs listed in Data S4. The resulting pools of PCR products were subjected to the third round of PCR (98°C x 3 min; 5 cycles of 98°C x 30 s, 60°C x 30 s, 72°C x 10 min; 72°C x 10 min; 4°C) to add Illumina P5 and P7 adaptor sequences using primers P5_sqF and P7_sqR (Data S4). The pools of adaptor-containing PCR products were sequenced for 110 cycles using NextSeq 500/550 Mid Output Kit