Genomic DNA isolation and amplification
Genomic DNA was isolated from the sorted cells using GenJet genomic DNA purification kit (Catalog #K0721). The sgRNA was amplified by PCR using Thermo Scientific Phire Green Hot Start II PCR master mix (Thermo Scientific, Catalog# F126S). For PCR amplification, genomic DNA samples were divided into 50uL PCR reaction. Each reaction consists of 25uL PCR master mix, 0.71uL of 14uM P5 stagger primer mix and 5uL of 14uM uniquely barcoded P7 primer mix, and rest of the volume was gDNA solution. All the P5 and P7 primers are shown in supplemental file. PCR cycling setting: initial denaturation 30 seconds at 98C; then 20 seconds at 98C, 15 seconds at 54C, 20 seconds at 72C for 25 cycles followed by 2 minutes extension at 72C. The pooled PCR products were run on a 2% agarose gel and correct band was extracted and purified with the Wizard SV gel and PCR clean-up system (Promega, catalog #A9281). All the sequencing primers are in Supplemental Table 1.