title: Tumor cell-intrinsic Decr2 is necessary for immunotherapy efficacy
design: Tumor cell-intrinsic Decr2 is necessary for immunotherapy efficacy
library_construction_protocol: To identify tumor cell-intrinsic genes essential for antigen-specific CD8+ T cell killing, we utilized the B16.SIY melanoma cell line which expressed the model antigen SIY (SIYRYYGL) co-expressed with DsRed, that can be recognized by 2C CD8+ TCR transgenic (Tg) T cells.  B16.SIY cells were transduced and selected to stably expressed a Cas9 gene, and then transduced with a genome-scale CRISPR gRNA library.  The library consists of 87,897 sgRNA sequences targeting 19,150 mouse protein-coding genes.  Transduced cells were cultured for 8 days to provide time for gene disruption to occur, then were co-cultured with pre-activated 2C CD8+ T cells at a 2:1 Effector:Target ratio for 16 hours, which we had previously shown results in around 99% killing of WT B16.SIY cells 27.  By performing deep sequencing, we examined the sgRNA library representation in tumor cells with or without T cell co-incubation by MAGeCK analysis Transduced cells were cultured for 8 days to provide time for gene disruption to occur, then were co-cultured with pre-activated 2C CD8+ T cells at a 2:1 Effector:Target ratio for 16 hours, which we had previously shown results in around 99% killing of WT B16.SIY cells The genomic DNA was extracted by MasterPure Complete DNA and RNA Purification Kit Cat No. MC85200 from LGC Biosearch Technologies two-step PCR amplification were performed on gDNA using  Q5Â® High-Fidelity DNA Polymerases (Neb). The first PCR step (PCR1) included amplification of the region containing sgRNA cassette using gRNA F1 (CTTGAAAGTATTTCGATTTC) and gRNA R (ACTCGGTGCCACTTTTTCAAGT) primers, and the second step PCR (PCR2) included amplification using uniquely Illumina barcoded adaptor-containing primers to allow multiplexing of samples in a single MiSeq.