SRCAP ChIP-sequencing of mouse intestinal stem cells (ISCs)

We previously demonstrated that SRCAP regulates the self-renewal of ESCs and modulates lymphoid lineage commitment. We further validated that SRCAP was mainly distributed in liver, spleen and intestine by Northern blot. SRCAP was also highly expressed in Lgr5+ ISCs. We then sought to explore the physiological role of SRCAP in the regulation of self-renewal maintenance of ISCs.We identified that Srcap deficiency impairs the self-renewal of ISCs and intestinal epithelial regeneration. Through SRCAP ChIP-sequencing, we sough to identify the key gene regulated by SRCAP in ISC self-renewal. Overall design: Primary Lgr5+ ISCs were isolated from tamoxifen treated Srcapflox/flox; Lgr5-Cre+ mice (Srcap KO) and Srcap+/+; Lgr5-Cre+ mice (Srcap WT). No in vitro culture were done. Crypts were isolated from Lgr5GFP mice.For ISC isolation, the crypts were dissociated into individual cells with 1x TrypLE express supplemented with 0.8 kU/ml DNaseI (Roche) followed by sorting according to GFP intensity. Sheared chromatin (sonicated to 200–500 bp) from ISCs (5×105) fixed in 1% formaldehyde was incubated with 4 µg antibody overnight at 4? followed by immunoprecipitation with salmon sperm DNA/protein agarose beads. After washing, elution and cross-link reversal, DNA from each ChIP sample and corresponding input sample were purified and prepared followed by library construction and sequenced on BGISEQ-500 platform (BGI America Co., Cambridge, MA).