GSM8851787: CScreen_Rapa_OCAB; Homo sapiens; OTHER
CRISPR screens: SU-DHL-4 reporter cells were transduced with the Brunello library. For each screen, 6 replicates with 15 million cells (90 million in total) were transduced at a density of 5 million cells/ml in a 6 well plate. The amount of virus was adjusted according to the viral titer to obtain a multiplicity of infection (MOI) of 0.3. Cells were transduced by spinfection (1,460g for 90 min and 30°C) in the presence of 6 µg/ml polybrene. Infected cells were puromycin selected 24h post infection at a final concentration of 4 µg/ml. On day 3 post infection, the MOI was verified and cells were split in medium containing 200 ng/ml doxycycline. Cells were split every other day with fresh doxycycline containing medium. On day 10 post infection, for each of the six replicates 60 million cells were sorted using BD FACS Aria or Fusion cell sorters based on reporter fluorescence to capture the bottom and top 5% expressing cells. On average, for each screen 286 million cells were sorted yielding ~9 million low or high expressing cells (Table S1B). For the three mTORC1 inhibited CRISPR screens in BTG2, CD79B and OCAB reporter cell lines, 5 nM rapamycin was added on day 8 and cells were sorted on day 10 as for the other screens. Genomic DNA from sorted cells was extracted using DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer's instructions. CRISPR screen Libraries were prepared using staggered GeCKO readout primers following the recommended instructions (Shalem et al. 2014). Deep sequencing was performed on NextSeq550 and NovaSeq6000 (Illumina) using 75bp single-end reads.