title: GSM4417274: SCNT_mESC_ctrl_H3K9me3; Mus musculus; ChIP-Seq
library_construction_protocol: The DNA extraction follows ChIP-seq protocol H3K27ac and H3K9me3 ChIP-seq in mESCs was performed as previously described (Brind'Amour et al., 2015; Liu et al., 2014). Briefly, mESCs were re-suspended in nuclear isolation buffer (Sigma). Depending on input size chromatin was fragmented for 5–7.5 min using MNase at 21 or 37 °C, and diluted in NChIP immunoprecipitation buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 15 mM NaCl, 0.1% Triton X-100, 1 × EDTA-free protease inhibitor cocktail and 1 mM phenylmethanesulfonyl fluoride (Sigma)). Chromatin was pre-cleared with 5 or 10 μl of 1:1 protein A: G Dynabeads (Life Technologies) and then incubated with antibody (H3K9me3, abcam, cat # ab8898; H3K27ac, Active Motif, cat # 39133) overnight at 4 °C. Next, complexes were washed twice with 400 μl of ChIP wash buffer I (20 mM Tris-HCl, pH 8.0, 0.1% SDS, 1% Triton X-100, 0.1% deoxycholate, 2 mM EDTA and 150 mM NaCl) and twice with 400 μl of ChIP wash buffer II (20 mM Tris-HCl (pH 8.0), 0.1% SDS, 1% Triton X-100, 0.1%deoxycholate, 2 mM EDTA and 500 mM NaCl). Protein–DNA complexes were eluted in 30 μl of ChIP elution buffer (100 mM NaHCO3 and 1% SDS) for 1.5h at 68 °C. DNA was purified by phenol chloroform and ethanol-precipitated. Purified DNA was subjected to Tru-seq library construction using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645S). The products were purified and size-selected with AMPure XP beads (Beckman Coulter, Cat # A63881).
attributes.tag: GEO Accession
attributes.value: GSM4417274