title: NextSeq 500 paired end sequencing; ATAC-seq of ESC or in vitro differentiated endoderm cells after epigenetic editing and upon release of the trigger
design: ATAC-seq of ESC or in vitro differentiated endoderm cells 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) 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 is to differentiate mESC into definitive endoderm with IDE1. The protocol is adjusted from Borowiak et al., Cell stem Cell., 2009. This ATAC-seq protocol was adapted from Corces et al. Nature Methods 2017. Briefly, fresh cells were collected and nuclei subjected to tagmentation with the Tn5 transposase using the Illumina Tagment DNA Enzyme and Buffer Kit #20034197. Fragmented DNA was isolated and subjected to library amplification with dual barcoded primers P5 and P7 Nextera XT Index kit (Illumina #15055293).