GSM8402774: GV_house_K4me3_1; Mus musculus; OTHER
Briefly, cells were pelleted and resuspended in nuclear isolation buffer (10 mM Tris-HCl, pH8.0, 140 mM NaCl, 5 mM MgCl2, 0.6% NP-40, 0.1% Triton X-100, 0.1% deoxycholate, 1 x EDTA-free proteinase inhibitor cocktail and 1 mM PMSF). Chromatin was then fragmented with MNase (2 U/μl at 37 °C for 7.5 min) and diluted in NChIP immunoprecipitation buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 150 mM NaCl, 0.1% Triton X-100, 1 × EDTA-free protease inhibitor cocktail and 0.1 mM PMSF). Carrier DNA (30 ng) was added into the fragmented chromatin to reduce DNA loss during immunoprecipitation. The fragmented chromatin was then pre-cleared with 5 μl of Dynabeads Protein A/Protein G (ThermoFisher) and immunoprecipitated overnight at 4 °C with the pre-prepared antibody-beads complex, which contains 700 ng H3K4me3 antibody (Digagenode, 15410003) or H3K27me3 antibody (Merk, 07‐449), H3K36me3 (Active motif, 61101) and 5 μl Dynabeads Protein A/Protein G. After immunoprecipitation, beads were washed twice with 150 μl of low salt wash buffer (20 mM Tris-HCl, pH 8.0, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 1 x EDTA-free proteinase inhibitor cocktail, 0.1 mM PMSF, and 0.2 ng/μl carrier DNA) and twice with 150 μl of high salt wash buffer (20 mM Tris-HCl, pH 8.0, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl, 1 x EDTA-free proteinase inhibitor cocktail, 0.1 mM PMSF, and 0.2 ng/μl carrier DNA). Protein-DNA complexes were eluted from beads in 100 μl ChIP elution buffer (100 mM NaHCO3 and 1% SDS) for 2 h at 65 °C. Immunoprecipitated DNA was purified by phenol-chloroform extraction followed by ethanol precipitation. Adaptor ligation was performed using the NEBNext Ultra II DNA Library Prep Kit for Illumina (E7645, New England Biolabs), and library pre-amplification was performed by 9 cycles of PCR. The pre-amplified library was digested by I-SceI (5 U/μl; 2 h incubation at 37 °C followed by 20 min heat inactivation at 65 °C). Barcoded final libraries were generated by further amplification of the carrier-DNA-removed pre-amplification products.