Deep mutational scan of TP53 in A549 cells

Isogenic p53 WT and p53 NULL A549 non-small cell lung cancer cells were stably infected with a pooled lentiviral ORF-based TP53 DMS library and treated with dimethyl sulfoxide (DMSO) vehicle, the MDM2 inhibitor nutlin-3, or the double-strand break-inducing chemotherapeutic agent etoposide for 12 days. Cells were collected just prior to treatment with drug or vehicle as well as at the end of the experiment. Proviral integrants were PCR-amplified from genomic DNA and subjected to sequencing and computational deconvolution with Analyze Saturation Mutagenesis v1.0 (ASMv1.0). Enrichment or depletion of single amino acid substitution variants was determined using the log2 fold-change in normalized reads between each day 12 sample and the corresponding early time point sample.