Genomic DNA (gDNA) was isolated using Blood & Cell Culture Mini Kits according to the manufacturer’s protocol (Qiagen). The extracted DNA and plasmid library were amplified as previously described12 (link). For each DNA sample from cells, we performed 30 separate 100 μl reactions with 7 μg genomic DNA in each reaction using KOD FX DNA Polymerase (TOYOBO) and then combined the resulting amplicons. Primer sequences to amplify the sgRNAs were Crispr seqF: 5’- TTGTGGAAAGGACGAAACACCG-3’ and Crispr seqR: 5’- TCTACTATTCTTTCCCCTGCACTGT-3’. The PCR products were purified using a gel extraction kit (Omega). Samples were sequenced on a NextSeq machine (Illumina) at BIOZERON Co., Ltd. (Shanghai, China). Reads were counted by first locating the CACCG sequence that appears in the vector 5’ in all gRNA inserts. The next 20 bases are the gRNA insert, which were then mapped to a reference file of all possible gRNAs present in the library using Bowtie 2.3.4.161 (link). Positive and negative selection genes were analyzed using MAGeCK software62 (link) with a threshold of p value < 0.05.
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