GSM3854818: MDP H3K27Ac; Mus musculus; ChIP-Seq
Sorted cells were cross-linked by incubating for 8 min at room temperature with 1% formaldehyde. Reactions were then quenched with 1.25 M glycine, cells were washed twice with PBS, and pellets were “flash frozen” for storage at -80°C. Chromatin was sonicated at 4°C in sonication buffer (10 mM Tris-HCl, pH 8.0; 100 nM NaCl; 1 mM EDTA, 0.5 mM EGTA; 0.1% sodium deoxycholate; and 0.5% N-lauroylsarcosine) for 24 cycles of 20 s on and 50 s off per cycle with a Vivra-Cell VCX130PB and CV188 (Sonics & Material) to obtain DNA fragments from 140 bp to 500 bp. Chromatin was then immunoprecipitated overnight at 4°C with Dynabeads Protein A that had been pre-incubated with 2.5 µg of the appropriate antibody: anti-H3K27ac (Ab 4729; Abcam) or H3K4me1 (Ab 8895; Abcam). Beads containing protein-DNA complexes were washed seven times with RIPA buffer (50 mM HEPES, pH 7.5; 500 mM LiCl; 1 mM EDTA; 1% NP-40; and 0.7% sodium deoxycholage). DNA fragments were eluted, and crosslinking was reversed by incubation in a solution containing 50 mM Tris-HCl, pH 8.0; 10 mM EDTA; 1% SDS; and 1 mg/mL of proteinase K (New England Biolabs) for 5 hours at 65°C. DNA was purified by phenol-chloroform extraction followed by ethanol precipitation. Libraries for ChIP-Seq were prepared with a ThruPLEX-FD kit (Rubicon Genomics)
GEO Accession
GSM3854818