Chromatin immunoprecipitation (ChIP)

Hdac1/2 DKO ES cells were processed for native ChIP-seq as previously described^44,45. Briefly, cell pellets were swollen in 0.3 M Sucrose, 60 mM KCl, 15 mM NaCl, 5 mM MgCl₂, 0.1 mM EGTA, 15 mM Tris-HCl, pH 7.5 and EDTA-free protease inhibitors (Sigma-Aldrich, UK). Nuclei were extracted using an equal volume of 0.3 M sucrose, 60 mM KCl, 15 mM NaCl, 5 mM MgCl₂, 0.1 mM EGTA, 15 mM Tris-HCl, pH 7.5, 0.4% NP-40, 0.5 mM DTT and protease inhibitors at 4 °C for 10 min. The cell suspension was applied to a sucrose cushion containing 1.2 M sucrose, 60 mM KCl, 15 mM NaCl, 5 mM MgCl₂, 0.1 mM EGTA, 15 mM Tris-HC pH 7.5 and centrifuge at 1500 rcf for 30 min at 4 °C. Nuclei were resuspended in MNase Digestion Buffer containing 0.32 M sucrose, 50 mM Tris-HCl, pH 7.5, 4 mM MgCl₂, 1 mM CaCl2 and protease inhibitors. Chromatin was then digested using 2UN/ml-1 Micrococcal nuclease (MNase) for 10–12 min at 37 °C on a thermal shaker. The reaction was stopped with 5 μM EDTA/EGTA and cleared at 12,000 rcf for 10 min at 4 °C. The supernatant (S1 fraction) was stored a 4 °C and the pellet was dialysed with 1 mM Tris-HCl, pH 7.5 and 0.2 mM EDTA overnight at 4 °C. The dialysed pellet (S2 fraction) was cleared at 20,000 rcf for 10 min at 4 °C 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 μL Dynabeads Protein G overnight at 4 °C. Bead were washed for 5 min in wash buffer A (50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 75 mM NaCl), wash buffer B (50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 125 mM NaCl), and wash buffer C (50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 175 mM NaCl). DNA was purified using IPure kit v2 (Diagenode, Belgium) according to the manufacturer’s instructions.