Genetic screening for single-cell variability modulators driving therapy resistance [WM989-A6-G3-Cas9 5a3]
We present the transcriptome of WM989-A6-G3-Cas9 5a3 melanoma cells followng the knockout (via CRISPR-Cas9) of different proteins we identified as modulators the frequency of cellular states associated with resistance to mutant-BRAF inhibition therapy. Overall design: We sequenced mRNA in bulk from WM989-A6-G3-Cas9 in order to quantify the transcriptional changes following the knockout of many targets from both the priming and resistance screens presented in Torre et. al. In addition to hits from our screens, we also quantified the transcriptome of targets that were not hits, but show a change in the frequency of NGFR-high/EGFRhigh cells or of cells resistant to vemurafenib. For each sample, we isolated mRNA and built sequencing libraries using the NEBNext Poly(A) mRNA Magnetic Isolation Module and NEBNext Ultra RNA Library Prep Kit for Illumina per manufacturer instructions. We then sequenced the libraries via paired-end sequencing (36x2 cycles) on a NextSeq 500. We aligned reads to hg19 and quantified reads per gene using STAR and HTSeq. Each sequenced sample represents the transcriptome resulting from the knockout with a single sgRNA design. On average, we targeted each protein with three sgRNA designs.