CRISPR-Cas9 Screening in MM1S and KMS18 Cells

MM1S and KMS18 cells were first virally transduced with LentiCas9-Blast (Addgene #52962) and Cas9 expression was confirmed by Western Blot (Cell Signaling Technology #14697). Subsequently, Cas9-positive cells were virally transduced with subpools of the Avana library of single guide RNAs (sgRNAs) in triplicate (~1K cells/guide), and samples were drawn on day 4 and day 27 following infection. DNA was extracted using QIAGEN DNA micro kits and deep targeted sequencing of the sgRNAs was performed at the Massachusetts General Hospital Center for Computational & Integrative Biology DNA Core. Following deconvolution and barcode quantitation with PoolQ, read counts were normalized for total depth, multiplied by 10^6, and log₂-transformed with a pseudocount of 1. Replicates with < 200K reads or correlation < 0.7 were discarded. The remaining technical replicates per subpool were collapsed by computing their median. Log₂ fold-changes were computed using day 4 as the baseline estimate and normalized by subtracting the median log₂ fold-change of all non-targeting sgRNAs in the respective subpool. A final guide-level matrix was obtained by computing the median log₂ fold-change across subpools. Copy number-corrected gene-level matrices were obtained using Ceres following z-mad normalization, as described previously, and used for downstream analyses^{57 (link)}.