EZH2 KO RNA-seq in HSC-5 cells

CRISPR was used to deplete EZH2 in HSC-5 cells. RNA-seq was then carried out to identify changes in the transcriptome following EZH2 loss in these cells. Critically, we identified TP63 to be downregulated in the EZH2-depleted cells, and also RUNX3 to be significantly upregulated. Overall design: Cas9 was stably expressed in HSC-5 cells via lentiviral transduction. The cells were then transduced with sgRNAs targeting EZH2. G418 was used to select for HSC-5 cells that had taken up the sgRNA. Following selection, EZH2 depletion was confirmed via western blotting and RNA was immediately collected for RNA-seq. 2 different sgRNAs were used. Samples were run in triplicate. One control was omitted from final analysis as it did not cluster with the others. Significantly changed genes were confirmed in follow up studies through RT-qPCR analysis.