A375 MHC-II+ screen Rep 2
A375-Cas9 and WM115-Cas9 were generated by transfection with lentivirus encoding Cas9-Blast (Addgene #52962) and selected with 6 g/mL and 12 g/mL blasticidin (InvivoGen #ant-bl-05) separately for 10 days. These two cell lines were independently transduced with Brunello lentivirus library (Addgene #73178) at an infection rate around 20%. sgRNA coverage was maintained throughout the experiments at > 500 copies of each sgRNA (~40 million cells for the ~77,000 sgRNAs). After 48 hours of transfection, cells that had been transduced were selected using 0.7 g/ml (A375) or 0.6 g/ml (WM115) of puromycin (InvivoGen #ant-pr-1) for 3 days. Following 13 days of viral transduction, the selected WM115 cells were treated with 100 ng/mL IFN- (Novoprotein #C014). Following 13 days of viral transfection, the cells were collected and assigned into three (for A375) or two (for WM115) replicates. Each replicate contained 56 million cells (~700x in sgRNA coverage) and were stained with the FITC-conjugated anti-HLA-DR antibody LN3 (Biolegend #327006), incubated on ice and protected from light for 20 min, washed with PBS, and resuspended with 2 mL PBS plus 5% fetal bovine serum prior to sorting for the lowest and highest ~10% populations, yielding ~2-4 million cells for each final population. The genomic DNA of the sorted samples and the control group was extracted using the NucleoSpin Blood XL kit (MACHEREY-NAGEL #740950.50), following the manufacturers instructions. Amplification of the sgRNA cassettes by PCR was performed according to the broad GPP protocol (https://portals.broadinstitute.org/gpp/public/resources/protocols).