Reference 2 library
Genome-wide CRISPR/Cas9 screens were performed using MC38 cells engineered to express Cas9 nuclease by lentiviral infection (pLentiCas9-Blast) and selection with blasticidin.  MC38-Cas9 cells were infected with the mouse GeCKO library (A and B combined) at a multiplicity of infection of 0.3 such that each sgRNA was introduced into ~200 cells.  Cells were selected with puromycin for 3 days and approximately 130 million cells were set aside as a reference control sample.   At 7 days post-infection, the T cell killing assay was set up in triplicate.  Library engineered cells at ~2000x library representation were pulsed with Ova peptide and cells at ~200x library representation were pulsed with control peptide.  After peptide pulsing, cells were co-cultured with activated CD8+ T cells (isolated from OT-1 mice) at an E:T ratio of 1:3.  After 24 hours of co-culture, when ~90% of the tumor cells had been killed, non-adherent cells were washed away with PBS and living tumor cells were harvested.  Genomic DNA extraction was subsequently performed using the DNeasy Blood & Tissue Kit (Qiagen) and NGS libraries were prepared as described previously (Joung et al., 2017).  The NGS libraries were subsequently multiplexed and run on the NextSeq 500 (Illumina), generating 80 base pair (bp) single-end reads. After demultiplexing with bcl2fastq software (Illumina), reads were screened for the 16 bp vector sequence adjacent to the sgRNA and the downstream 20 bp sgRNA reads were extracted for the sgRNA count.