title: GSM3207245: dKO_Input_rep2; Mus musculus; ChIP-Seq
library_construction_protocol: Briefly, cell pellets were swollen in 0.3M Sucrose, 60mM KCl, 15mM NaCl, 5mM MgCl2, 0.1mM EGTA, 15mM Tris-HCl, pH 7.5 and EDTA-free protease inhibitors (Sigma-Aldrich, UK). Nuclei were extracted using an equal volume of 0.3M sucrose, 60 mM KCl, 15mM NaCl, 5mM MgCl2, 0.1mM EGTA, 15mM Tris-HCl, pH 7.5, 0.4% NP-40, 0.5 mM DTT and protease inhibitors at 4C for 10 min. The cell suspension was applied to a sucrose cushion containing 1.2M sucrose, 60 mM KCl, 15mM NaCl, 5mM MgCl2, 0.1mM EGTA, 15 mM Tris-HC pH 7.5 and centrifuge at 1500 rcf for 30min at 4C. Nuclei were resuspended in MNase Digestion Buffer containing 0.32M sucrose, 50mM Tris-HCl, pH 7.5, 4mM MgCl2, 1mM CaCl2 and protease inhibitors. Chromatin was then digested using 2UN/ml-1 Micrococcal nuclease (MNase) for 10-12min at 37C on a thermal shaker. The reaction was stopped with 5 μM EDTA/EGTA and cleared at 20,000 rcf for 10 min at 4C. The supernatant (S1 fraction) was stored a 4C and the pellet was dialysed with 1mM Tris-HCl, pH 7.5 and 0.2mM EDTA overnight at 4C. The dialysed pellet (S2 fraction) was cleared at 20,000 rcf for 10 min at 4C and combined with S1 fraction for immunoprecipitation. Chromatin was immunoprecipitated using 8 μg H3K18ac (Catalog No: 39755; Active Motif) or 8 μg H3K18cr (PTM-Biolabs 517) conjugated to 50 uL Dynabeads Protein G overnight at 4C. Bead were washed for 5min in wash buffer A (50mM Tris-HCl, pH 7.5, 10mM EDTA, 75mM NaCl), wash buffer B (50mM Tris-HCl, pH 7.5, 10mM EDTA, 125mM NaCl), and wash buffer C (50mM Tris-HCl, pH 7.5, 10mM EDTA, 175mM NaCl). DNA was purified using IPure kit v2 (Diagenode, Belgium) according to the manufacturer's instructions. For next-generation sequencing, automated library preparation were prepared from 10 ng of ChIP and input DNA 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 (Oxford Genomics Centre) dual indexing primers (based on DOI: 10.1186/1472-6750-13-104).