GSM5859187: Wild Type Mouse Embryonic Fibroblast, rep1; Mus musculus; ChIP-Seq
Cells were crosslinked (two biological replicates per condition and input controls) using 1% formaldehyde (Sigma Cat. No. F8775) for 10 min followed by quenching with 0.125 M Glycine for 5 min and lysis with lysis buffer 1-LB1-(50 mM Hepes KOH pH 7.5, 10 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100). Nuclei were pelleted, collected and washed using lysis buffer 2-LB2- (10 mM Tris-HCl pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA). This was followed by lysis using lysis buffer 3 LB3 (10 mM Tris-HCl pH 8; 100 mM NaCl, 1 mM EDTA; 0.5 mM EGTA; 0.1% Na-Deoxycholate, 0.5% N-laurylsarcosine). Chromatin was sheared using Qsonica Sonicator (4 cycles of 3 min at 70 % Amplitude), and then checked on 0.8% agarose gel. 100 μg of fragmented chromatin was mixed with 5 μg of H3K27ac-Anti-Histone H3 (acetyl K27) antibody-ChIP Grade (ab4729) - abcam). The protein-antibody immunocomplexes were recovered by the Pierce Protein A/G Magnetic Beads (Thermo-Scientific). Beads and attached immunocomplexes were washed twice using Low salt wash buffer (LS) (0.1% SDS; 2 mM EDTA, 1% Triton X-100, 20 mM Tris-HCl pH 8, 150 mM NaCl), and High Salt (HS) wash buffer (0.1% SDS, 2 mM EDTA, 1% Triton X-100, 20 mM Tris-HCl pH 8, 500 mM NaCl), respectively. The beads were resuspended in elution buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, 1% SDS). De-crosslinking was achieved by adding 8 µL 5 M NaCl and incubating at 65 °C overnight. RNase A (1 µL 10 mg/mL) was added for a 30 min incubation at 37 °C. Then, 4 µL 0.5 M EDTA, 8 µL 1 M Tris-HCl, and 1 µL 20 mg/mL proteinase K (0.2 mg/mL) were added for a 2-hour incubation at 42 °C to digest the chromatin. DNA was purified by QIAquick PCR purification kit (Qiagen, Germantown, MD, USA) for qPCR analysis and sequencing. Library preparation and sequencing was performed by Novogene Co., Ltd using standard illumina protocols. Library preperation and sequencing was performed by Novogene. Co Ltd using standard illumina protocols