title: NextSeq 500 sequencing; CRISPR screen to identify regulators of epigenetic memory in ESC
design: CRISPR screen to identify regulators of epigenetic memory in ESC
library_construction_protocol: Esg1-tdTomato or p53-tdTomato reporter ESC line containing inducible dCas9::KRAB or dCas9::GFP control and target-specific gRNA have bee treated with DOX (100 ng/ml) for 7 days to impose epigenetic silencing of the reporter. Memory of the silenced state has been assayed after 4 or 7 days of DOX washout. For the screen, ESC line carrying a spCas9-GFP and the endogenous Esg1-tdTomato reporter have been transduced with lentiviral gRNA library (Doench et al., 2016) and selected with puromycin. After inactivation of the SpCas9-GFP cells have been transfected with the epigenetic editing tool to impose silencing of the Esg1-tdTomato reporter for 7 days by DOX induction. tdTomato negative cells were sorted at FACS and put back in culture in absence of DOX for other 3 or 7 days. At this timepoints cells were sorted again using 3 gating strategy to separate the 2,5% bottom tdTomato negative cells or cells ranging from fully to mildly silenced (TOMneg-wide) and in parallel also tdTomato positive cells were isolated. These cells were then subjected to DNA extraction using DNeasy blood and tissue kit (Qiagen # 69504) following manufacturer instruction including RNAse step. Genomic DNA extracted from sorted cells was subjected to library amplification with custom primers containing the P7 flow cell overhangs using the Q5 Hot Start High-Fidelity polymerase (NEB #M0494S) for 22-24 cycles. sgRNA amplicons were purified with SPRI beads, checked and quantitated with a tape station automated electrophoresis system and pooled for next generation sequencing.