Next Generation Sequencing and Bioinformatics

DNA samples obtained from the CRISPR-Cas9 GeCKO screen were used to prepare a sgRNA library by a two-step PCR as described before (40, 41). The first PCR was used to amplify the sgRNA-containing cassette. Resulting PCR products were subjected to a second PCR using a primer pair encoding a unique 8-bp barcode required for multiplexing along with a stagger sequence to increase library complexity as described elsewhere: http://sanjanalab.org/lib.html (40, 41). Purified PCR products of the second PCR were pooled, diluted, and mixed with 10% PhiX, and sequenced with a MiSeq machine using a Micro Kit V2 flowcell (300 cycles). The raw sequencing data were processed and analyzed using the CRI CRISPR-Cas9 library screen pipelines according to MAGeCK v0.5.8 (42). In brief, sequencing reads were first de-multiplexed by using the barcode in the reverse primer and processed by Cutadapt v2.10 (43) to remove potential adapter sequences from the beginning of sgRNA priming site primers. Read counts of sgRNAs for each sample were quantified by applying MAGeCK’s count command. Subsequently, samples were compared using MAGeCK’s test command and genes were ranked according to their score. Visualization of results were done in R v4.1.0 using ggplot2 (44).