GSM5550100: KO H3K27ac ChIP; Mus musculus; ChIP-Seq
2-4x107 cells were cross-linked for 10 minutes at room temperature by the addition of formaldehyde solution at final concentration of 1% followed by quenching with glycine. Cells were washed twice with PBS, then the supernatant was aspirated and the cell pellet was conserved in -80℃. Pellets were lysed, resuspended in 1mL of LB1 on ice for 10 min (50 mM HEPES-KOH pH 7.4, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10% Glycerol, 0.5% NP40, 0.25% Tx100, protease inhibitors), then after centrifugation resuspend in LB2 on ice for 10 min (10 mM Tris pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA and protease inhibitors). After centrifugation, resuspend in LB3 (10 mM Tris pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% NaDOC, 0.1% SDS andprotease inhibitors) for histone marks and SDS shearing buffer (10mMTris pH8, EDTA 1mM, SDS 0.15% and protease inhibitors) for transcription factor and sonicated (Covaris settings: 5% duty, 200 cycle, 140 PIP, 20 min), yielding genomic DNA fragments with a bulk size of 100-300bp. Coating of the beads with the specific antibody and carried out during the day at 4℃, then chromatin was added overnight at 4℃ for histone marks while antibody for transcription factor is incubated with chromatin first with 1% Triton and 150mM NaCl. Subsequently, washes were performed with 2x Low Salt Wash Buffer (10 mM Tris pH 8, 1 mM EDTA, 150mM NaCl, 0.15% SDS), 1x High Salt Wash Buffer (10 mM Tris pH 8, 1 mM EDTA, 500 mM NaCl, 0.15% SDS), 1x LiCl buffer (10 mM Tris pH 8, 1 mM EDTA, 0.5 mM EGTA, 250 mM LiCl, 1% NP40, 1% NaDOC) and 1 with TE buffer. Final DNA was purified with QIAGEN Elute Column. Up to 10 nanograms of ChIPed DNA or input DNA (Input) were prepared for sequencing. We performed library construction using the KAPA Hyper Prep Kit (Roche)