title: GSM8128096: H3K27ac ChIP-seq in ESC after NVP-2 treatment for 120min treatment rep2; Mus musculus; ChIP-Seq
library_construction_protocol: ChIP assay was performed as described previously (Narita et al., 2023) with modifications. mESCs were treated two hours with DMSO, 1 μg/ml Actinomycin D (Tocris Bioscience), or 100 nM NVP2 (Tocris Bioscience), respectively. After treatments, cells were cross-linked with 1% formaldehyde for 10 minutes, neutralized with 0.2 M glycine for 5 minutes, washed twice with PBS, then collected by cell lifter, concentrated as pellets by centrifuged for 10 min at 3000 rpm at 4 °C and stored at -80 °C until use. Cells were resuspended in SDS lysis buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% SDS, 1 mM EDTA, pH 8.0, cOmplete Protease Inhibitor Cocktail (Roche)) and sonicated by a Bioruptor Pico (30 cycles, 30 seconds on and 30 seconds off, Diagenode), then centrifuged for 10 min at 13,000×g at 20 °C for debris removal. The supernatants were stored at -80 °C. The aliquots of the supernatant were 5-fold diluted with ChIP dilution buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, cOmplete Protease Inhibitor Cocktail), incubated 20 hours at 65 °C to reverse cross-linking, then treated with 0.2 mg/ml RNase A (Thermo Fisher Scientific) for 30 min at 37 °C and proteinase K (Thermo Fisher Scientific) for 1 hour at 55 °C. Then DNA fragments were isolated by phenol-chloroform extraction and recovered by ethanol precipitation with glycogen (Thermo Fisher Scientific). DNA fragments were quantified by Nanodrop (Thermo Fisher Scientific). The 300 bp average size of DNA was measured by 4200 TapeStation System (Agilent Technologies). For immunoprecipitation, the supernatants equivalent to 100 μg of mESC chromatin DNA combined with 10 μg of chromatin DNA derived from HEK293T as spike-in were incubated with anti-histone H3 acetyl K27 antibody (ab4729 Abcam) conjugated with Dynabeads M-280 sheep anti-Rabbit IgG (Thermo Fisher Scientific) by rotating overnight 4 °C . The immunoprecipitated Protein-DNA complexes were washed with ChIP dilution buffer, wash buffer high-salt (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), wash buffer low-salt (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% sodium deoxycholate, 0.5% NP-40) once respectively, washed with TE three times, then eluted with elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS). The immunoprecipitated DNAs were reversed cross-link and recovered from the eluates as described above. ChIP-seq, library was prepared from 20 ng of ChIPed DNA measured by Qubit dsDNA HS Assay (Thermo Fisher Scientific) using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer's instructions. Adoptor-ligated, U-excisioned DNA was cleaned by AMPure XP beads (Beckman Coulter) amplified by PCR 6 cycles, then cleaned again and size-selectioned (350-400 bp average size measured by Tapestation). 4 nM pooled Library DNAs were sequenced on a NextSeq 2000 Sequencer (Illumina) as single-end 138-bp reads.