Homo sapiens Epigenomics

Cas9 expressing HUDEP-2 cells in expansion media were transduced in parallel with sgRNAs targeting CHD4 and MTA2 and controls. 24 hours following transduction, media was replaced with fresh expansion media containing 1µg ml-1 puromycin. After 48 hours of transduction, a small fraction of puromycin selected cells was used to extract genomic DNA. Four days following transduction cells were lysed and RNA was extracted using RNeasy Mini Kit (Qiagen). Libraries were synthesized using Illumina TruSeq Stranded mRNA sample preparation kits from 500 ng of purified total RNA according to the manufacturer''s protocol. The final dsDNA libraries were quantified by Qubit fluorometer, Agilent TapeStation 2200, and RT-qPCR using the Kappa Biosystems library quantification kit according to manufacturer''s protocols. Uniquely indexed libraries were pooled in equimolar ratios and sequenced on an Illumina NextSeq500 with single-end 75bp reads by the Dana-Farber Cancer Institute Molecular Biology Core Facilities.