title: GSM3892052: HN12_DMSO_rep1; Homo sapiens; OTHER
library_construction_protocol: Total genomic DNA was extracted with the Quick-gDNA MiniPrep Kit (Zymo Research Corporation) according to manufacturer's instructions. Synthetic Lethal Screen : 4x10^6 cells from each of the cell lines (UMSCC8, UMSCC25, HN12, Cal27, JHU011) were plated into duplicate 15cm plates (3x10^6 cells/plate) and transduced 24 hours later with the kinome shRNA library containing viral supernatant and 8 μg/ml polybrene (Sigma-Aldrich). After 72 hours, transfected cells were selected with puromycin (1 μg/ml) for 5 days. The lentiviral preparation for the SBI genome-wide screen was reported in our previous publication Essential Kinome Screen : Target cell lines (UMSCC8, UMSCC25, HN6, HN12, Cal27, JHU011) were plated at 5x10^6 cells per 15 cm plate, 2 plates per cell line, in 20 mL growth medium. The following day, cells were pretreated with 1 µg/mL polybrene for 30 minutes. The media containing the packaged lentiviral kinome shRNA library was added to each plate of target cells at a MOI of ~0.2, incubated for 16 hours and then removed and replaced with fresh growth medium (lacking puromycin). Forty-eight hours post-infection, each cell line was trypsinized and plated at a density of at least 2x10^6 cells per 15 cm plate in quadruplicate. Additionally, at the same 6 time point, four replicates of 4x10^6 cells each from each cell line were pelleted.