GSM7103847: K27ac_D5F_ABV_Input; Mus musculus; ChIP-Seq
Cells sorted at Day 4 or Day 5 of differentiation and frozen with liquid nitrogen were crosslinked in 0.5% formaldehyde for 5 min at room temperature, and then the reaction was quenched with 125 mM glycine. Cross-linked cells were rinsed with PBST (PBS with 0.1% Tween 20) and then resuspended in LB3-Triton (1 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl pH 8, 100 mM NaCl, 0.1% Na-Deoxycholate, 0.5% N-lauroyl sarcosine, 1% Triton) supplemented with a protease inhibitor cocktail (Calbiochem 539131). Chromatin was extracted by sonication using Bioruptor (Diagenode). Cell extract was cleared by centrifugation and an aliquot was saved for input DNA sequencing. Cell extract from 0.5 million cells was incubated with mouse monoclonal anti-H3K27ac antibody (Wako MABI0309, Lot 14007; 2 μL per IP) in a 200 μL reaction for 12 hours or longer at 4oC. Immunocomplex was captured by Protein G-conjugated magnetic dynabeads (ThermoFisher) and washed. Immunoprecipitated DNA was reverse-crosslinked and used to construct high-throughput sequencing libraries using NEBNext Ultra II DNA Library Prep Kit (New England Biolabs). DNA libraries were processed on an Illumina HiSeq machine for paired-end sequencing.