Whole-genome CRISPR screen of etoposide-treated U2OS and U2OS p53KO cells, and RNAseq of U2OS cells incubated in media conditioned by etoposide-treated U2OS or U2OS p53KO cells.
CRISPR screen: U2OS or U2OS p53KO cells expressing Cas9 were transduced with a whole-genome library of CRISPR sgRNAs, then treated with either DMSO or etoposide. Differential sgRNA abundances were calculated for each condition and used to determine the effect of each single gene knockout on fitness and the drug-induced death rate. RNA-Seq: U2OS or U2OS p53KO cells were cultured with either DMSO or etoposide for 48 hours, and then U2OS cells were incubated in this conditioned media for 8 hours. RNA was collected and use to observe differential expression changes between conditions. Overall design: CRISPR screen: Pooled library of U2OS or U2OS p53KO cells was treated with DMSO or etoposide for 96 hours, gDNA was isolated using phenol-chloroform extraction, sgRNA libraries were amplified and barcoded using PCR, whole-genome libraries were sequenced on an Illumina HiSeq platform. RNA-Seq: U2OS cells were cultured in media conditioned by U2OS or U2OS p53KO cells for 8 hours, RNA was extracted using a Qiagen RNeasy Mini Kit, total RNA was sequenced by BGI Genomics.