CRISPR Screening of Head and Neck Cancer

Cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen #11965) containing 10% fetal bovine serum (FBS, Sigma), 1% NEAA (Invitrogen 15140122) and 7 μL/mL penicillin-streptomycin (Invitrogen 15140122) in a humidified atmosphere of 5% CO₂ at 37°C, as described [17 (link)]. Cells were tested for mycoplasma contamination using the MycoAlert detection kit (Lonza) and genotyped, as described [18 (link)]. UM-SCC lines were transduced with the Human Genome-wide CRISPR KnockOut (GeCKO) pooled library, version 2A which was a gift from Feng Zhang [15 (link)]. Conditions for transduction were established for a multiplicity of infection (MOI) of 30%. After 7 days of puromycin selection, the cells were expanded and seeded per treatment. To preserve at least 300x coverage, 30 million cells were seeded per treatment for the GeCKO libraries. At the end of treatment, DNA was extracted from the remaining cells using Gentra Puregene Cell Kit (Qiagen), as described [19 (link)]. For cisplatin treatment, cells were dosed with 0.125 μM cisplatin (Selleckchem S1166) or DMSO (Sigma Aldrich) for 24 hours, once a week for two weeks.