title: GSM5733535: 130kb RARE∆ SynHoxA assemblon in HoxA -/- mESCs, 24h of RA+SAG differentiation, replicate 1, H3K27me3 ChIP; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were collected at 0h or 24h, 48h, or 96h after RA/SAG treatment. Cells were crosslinked at room temperature in 1 mM DSG (ProteoChem) for 15 min, followed by the addition of 1% FA (vol/vol) for 15 min. The reaction was quenched with Glycine and cells were washed with 1 × PBS. Samples were divided into ~25-30x106 cell aliquots, pelleted by centrifugation at 275 g, and frozen at -80°C. Cell aliquots were thawed on ice and lysis was performed in 5 mL of 50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8.0, 10% glycerol (vol/vol), 0.5% Igepal (vol/vol), 0.25% Triton X-100 (vol/vol) with 1 × protease inhibitors (Roche, 11697498001) for 10 min at 4°C. Cells were pelleted by centrifugation for 5 min at 1200 g, resuspended in 5 mL of 10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0 with 1 ×