Genome-scale CRISPR/Cas9 Knockout Screening

Q5 Hot Start High-Fidelity (NEB) was used to amplify the DNA fragments with 300 bp covering the guide sequence, and the fragments were purified by a Precellys Ceramic Kit (Peqlab). Different paired barcodes were ligated to the fragment by a secondary round PCR in each tumor. All the barcoded samples were pooled to be sequenced. The sequenced raw data were demultiplexed and trimmed to be 20 nt sgRNA sequences, followed by mapping with sgRNA sequences in the CRISPR knockout library. MAGeCK 23 (link) was used to identify positively and negatively selected sgRNAs and genes in genome-scale CRISPR/Cas9 knockout experiments. The method consists of read count normalization, sgRNA ranking and gene ranking, and the enriched sgRNAs are shown in a ranked list.