Early timepoint and late timepoint triple replicates for MCF7, HELA, HA1E, HEPG2
The lentiviral barcoded library used in the primary screen contains 5235 sgRNAs, which includes an average of 8 guides per gene and 500 non targeting control guides. The validation library contains 6996 sgRNAs targeting selected regions of the smORFs. Genome-scale infections were performed in three replicates with the pre-determined volume of virus in the same 12-well format as the viral titration described above, and pooled 24 h post-centrifugation. Infections were performed with enough cells per replicate, in order to achieve a representation of at least 1000 cells per sgRNA following puromycin selection (~1.5x10e7 surviving cells). Approximately 24 hours after infection, all wells within a replicate were pooled and were split into T225 flasks. 24 hours after infection, cells were selected with puromycin for 7 days to remove uninfected cells. After selection was complete, 1.5-2x10e7 of cells were harvested for assessing the initial abundance of the library. Cells were passaged every 3-4 days and harvested ~21 days after infection. For all genome-wide screens, genomic DNA (gDNA) was isolated using Midi or Maxi kits for the validation screens gDNA was isolated using and Midi kits according to the manufacturer's protocol (Qiagen, Hilden, Germany). PCR and sequencing were performed as previously described.  Samples were sequenced on a HiSeq2000 (Illumina, San Diego, CA). For analysis, the read counts were normalized to reads per million and then log2 transformed. The log2 fold-change of each sgRNA was determined relative to the initial time point for each biological replicate.
SUB8622122
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