GSM4174031: H3K27ac_veh, biological rep1; Mus musculus; ChIP-Seq
Cells were crosslinked in 1% formaldehyde for 10 min at room temperature followed by addition of glycine (125 mM final concentration) for 5 min. Nuclei were isolated using lysis buffer 1 (5 mM Pipes pH 8, 85 mM KCl, 0.5% NP40, and complete protease inhibitor cocktail (Roche)) and were lysed using lysis buffer 2 (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1, and complete protease inhibitor cocktail (Roche)). Chromatin was sheared into an average size of 500 bp by 8 s pulses (30 s pause between pulses) at 4 °C in the water bath sonicator Bioruptor (Diagenode, Liège, Belgium). Thirty micrograms of chromatin were incubated overnight at 4 °C in rotation, diluted 1:10 in IP buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, and 167 mM NaCl, 1% sodium deoxycholate) and with the respective antibodies: 3 µg of anti-H3K27ac (Ab4729, Abcam.), 3 µg of anti-H3K4me1 (Ab8895, Abcam) and 3 µg of anti-K3m4me3 (Ab8580, Abcam). Immunoprecipitations were incubated for 2 h at 4 °C in rotation with protein A Dynabeads (Invitrogen) and then washed with wash buffer 1 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, and 150 mM NaCl, 1% sodium deoxycholate), wash buffer 2 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, and 500 mM NaCl, 1% sodium deoxycholate), wash buffer 3 (0.25 M LiCl, 1% NP40, 1% sodium deoxycholate, 1 mM EDTA, and 10 mM Tris-HCl pH 8.1), and twice with TE buffer (10 mM Tris-HCl pH 8.0 and 1 mM EDTA). The complexes were eluted from the beads with elution buffer (1% SDS in TE buffer) by incubating twice 10 min at 65 °C. The eluates and the inputs were incubated overnight at 65 °C for de-crosslinking and chipped DNA was purified with phenol:chloroform extraction followed by ethanol precipitation and resuspended in miliQ water
GEO
GSM4174031
GEO Accession
GSM4174031