GSM3772697: TKO.DMSO_H3ac_REP1; Mus musculus; ChIP-Seq
Chicken DF-1 and mouse ES cells were harvested by trypsinisation. For each experimental condition, 5 x 106 chicken DF-1 cells were added to 5 x 107 mESCs. Cell mixtures were pelleted and resuspended in 1 ml ice-cold RSB Buffer (10 mM NaCl, 10 mM Tris (pH 8.0), 3 mM MgCl2). Chromatin was fragmented by adding 200 units of Micrococcal Nuclease (MNase, Fermentas) and incubating at 37 °C for 5 min. The reaction was stopped by the addition of 8 µl of 0.5 M EDTA and spun at 2348 x g for 5 min at 4 °C. The supernatant containing soluble nucleosomes was collected (S1 fraction). Nucleosomes in the remaining insoluble fraction were extracted by rotating in 300 µl Nucleosome Release Buffer (10 mM Tris pH 7.5, 10 mM NaCl, 0.2 mM EDTA, with 1x cOmpleteTM EDTA-free Protease Inhibitor Cocktail (Roche) and 10 mM sodium butyrate) for 1 h at 4 °C followed by the passing through a 25G needle five times. The supernatant was collected as described above and pooled with the S1 chromatin fraction.   Immunoprecipitations were set up with 100 µl solubilised chromatin and 3 µg of an anti-acetylated histone H3 antibody in a total of 1 ml Native ChIP Incubation Buffer (10 mM Tris pH 7.5, 70 mM NaCl, 2 mM MgCl2, 2 mM EDTA and 0.1% Triton X-100, 1x cOmpleteTM EDTA-free Protease Inhibitor Cocktail and 10 mM sodium butyrate) and rotated overnight at 4 °C. Immune complexes were captured using 20 µl Protein A agarose beads (Roche, previously saturated with 1 mg/ml BSA and 1 mg/ml yeast tRNA) for 1 hour at 4°C with rotation, washed four times with Native ChIP Wash Buffer (20 mM Tris pH 7.5, 2 mM EDTA, 125 mM NaCl, 0.1% Triton-X100) and once in TE buffer (10 mM Tris HCl pH 8.0, 1 mM EDTA) and subsequently eluted in 100 µl elution buffer (1% SDS, 0.1 M NaHCO3) by vigorous shaking for 30 min at room temperature. DNA was purified using the ZymoResearch ChIP DNA clean and concentrator kit (Zymo Research) following the manufacturer's instructions. For input samples, DNA was purified from 100 µl of solubilised chromatin using the same DNA purification procedure. Automated library preparation was performed on 5 ng input material using the Apollo prep system (Wafergen, PrepX ILMN 32i, 96 sample kit) and standard Illumina multiplexing adapters following manufacturer's protocol up to pre-PCR amplification. Libraries were PCR amplified (18 cycles) on a Tetrad (Bio-Rad) using the NEBNext High-Fidelity 2X PCR Master Mix (NEB) and in-house single indexing primers. Equivalent amounts of individual libraries were pooled together. Paired-end sequencing was performed using a HiSeq4000 75 bp platform (Illumina, HiSeq 3000/4000 PE Cluster Kit and 150 cycle SBS Kit), generating a raw read count of >30 million reads per sample.