GSM2610613: ws998M18_H3K27ac_ChIPSeq; Homo sapiens; ChIP-Seq
Chromatin preparation was performed using the NEXSON protocol (doi:10.1093/nar/gkv1495) with some modifications. Up to and including immunoprecipitation all buffers were supplemented with Complete Protease Inhibitor Cocktail (Roche) and 10 mM Sodiumbutyrate (Sigma-Aldrich, St. Louis, USA). Briefly, fixed cells were resuspended in 1 ml Farnham lab buffer (5 mM PIPES pH 8, 85 mM KCl, 0.1% Igepal CA-630) and sonicated in a Bioruptor NGS (Diagenode) with 3 cycles (15 s on and 30 s off) in high power mode using standard 1.5 ml tubes. After centrifugation nuclei were resuspended in shearing buffer (10 mM Tris-HCl (pH 8), 0.1% SDS, 1 mM EDTA) and chromatin was sonicated into fragments between 100 and 800 bp using 1.5 ml microtubes with caps (Diagenode, C30010016) for 6 cycles (30 s on and 30 s off) in high power mode. For each immunoprecipitation 10 µl protein G beads (Invitrogen) were combined either with 2 μg H3K4me3 (Diagenode, C15410003-50) or H3K27ac antibody (C15410196) and 5 μl BSA (NEB, 10 mg / ml) and rotated for 2 h at 4 °C. Then, 80 µl sheared chromatin (equal to approx. 106 cells) diluted with double volume dilution buffer (25 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 % Triton X-100, 0.75 % Glycerol) were added. After immunoprecipitation overnight beads were consecutively washed with 1 ml of WB1 (20 mM Tris-HCl, (pH 8.0), 150 mM NaCl, 2 mM EDTA, 1 % Triton X-100, 0.25 % SDS), twice WB2 (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 2 mM EDTA, 1 % Triton X-100, 0.25 % SDS), WB3 (10 mM Tris-HCl, (pH 8.0), 250 mM LiCl, 1 mM EDTA, 0.5 % Nonident P-40, 0.5 % Sodiumdesoxycholate) and twice Tris Buffer  (10 mM Tris-HCl (pH 8.0)) under rotation for 10 min at 4 °C. Library construction was performed as previously described (doi:10.1038/nmeth.3542) with modifications. In brief, immunoprecipitated chromatin bound to beads was incubated in 25 µl tagmentation mix containing 1 µl Nextera Tn5 (Illumina, FC-121-1030) for 10 minutes at 37 °C. Beads were then washed twice with WB1 and TE/NaCl (10 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1 mM EDTA) and eluted in 70 µl elution buffer (10 mM Tris-HCl (pH 8), 300 mM NaCl, 5 mM EDTA, 0.5 % SDS) containing 2 µl proteinase K (Sigma-Aldrich, P6556) over night at 65 °C. After magnetic separation eluates were transferred into fresh tubes, while the beads were resuspended in additional 30 µl elution buffer and incubated for 10 minutes at 65 °C. Eluates were combined, supplemented with 1 µl proteinase K and incubated for 1 hour at 55 °C. Tagmented DNA was purified using the MinElute PCR Purification Kit (Qiagen, 28004) and amplified using 1 X NEBNext® High-Fidelity PCR Master Mix (NEB, M0541S) and 0.2 µM of Nextera PCR primer 1 (AATGATACGGCGACCACCGAGATCTACACTCG TCGGCAGCGTCAGATGTG) and indexed primer 2 (CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTCTCGTGGGCTC GGAGATGT) in a 50 µl reaction with the following PCR conditions: 72 °C for 5 min, 98 °C for 30 s, 12 cycles of 98 °C for 10 s, 63 °C for 30 s, 72 °C for 1 min and a final elongation at 72 °C for 7 min. Final library clean-up was performed with 1 X AMPure XP beads (Beckman Coulter). Libraries were sequenced 2x 50 bp using a v3 paired-end flowcell on a HiSeq2500 system (Illumina).