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.