title: NextSeq 500 paired end sequencing; CUT&RUN-seq of H3K9me3, H4K20me3 or H3K4me3 in mESC after epigenetic editing and upon release of the trigger
design: CUT&RUN-seq of H3K9me3, H4K20me3 or H3K4me3 in mESC after epigenetic editing and upon release of the trigger
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. This protocol has been optimised from Skene and Henikoff, Elife, 2017 to detect interaction between DNA and histone modifications. Briefly, fresh cells are harvested, permeabilised, bound to concanavallin magnetic beads and incubated with the antibody for the modification of interest. Samples were then incubated with pA-MNase to cut the DNA side of the bound antibody and the DNA fragments were eluted and subjected to size selection. Finally DNA library was generated with NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB #E7645S).