GSM5017144: H3_histone_D458_with_DMSO; Homo sapiens; ChIP-Seq
The growth medium was aspirated from cells (70% confluence) and replaced with a 1% formaldehyde in 1X DPBS for 10 min at room temperature. The cross-linking was terminated by addition of glycine to a final concentration of 0.125 M and incubation for 5 min. The cells were washed twice with cold PBS and scraped in ChIP lysis buffer (1% SDS, 10 mM EDTA and 50 mM Tris-HCl pH 8.1) containing COmplete EDTA free protease inhibitors (Sigma). The cell lysates were sonicated with a Bioruptor Plus (Diagenode) for 25 cycles (30 seconds ON and 30 seconds OFF). The size of the sonicated DNA fragments was checked on an 1X TAE1 % agarose gel electrophoresis and ranged between 250 to 500 bp. After removing the debris via centrifugation, chromatin extracts were collected and protein concentration was determined by BCA protein assay reagents (Pierce).  For immunoprecipitation, chromatin extracts with 1 mg of total protein for each sample were incubated with primary antibodies (dilution 1:50) overnight at 4ºC. Primary antibodies used are included in table under “Western blotting” below. After incubation with the primary antibody, 20 uL of pre-washed magnetic beads (Magna ChIP Protein A+G Magnetic Beads, Millipore Sigma) were added to each sample for 2 hours at 4ºC. Using a magnetic rack, the immunoprecipitates were washed successively with 1 ml of low salt buffer (20 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% SDS, 1% triton X-100, 2 mM EDTA), high salt buffer (20 mM Tris-HCl [pH 8.0], 500 mM NaCl, 0.1% SDS, 1% triton X-100, 2 mM EDTA), LiCl washing buffer (10 mM Tris-HCl [pH 8.0], 250 mM LiCl, , 1.0% NP40, 1.0% deoxycholate, 1 mM EDTA) and twice with TE buffer. The DNA-protein complexes were eluted with 300 μl of IP elution buffer (1% SDS, 0.1 M NaHCO3). The eluents were pooled together and the cross-links were reversed by adding NaCl (a final concentration 0.2 M) into the eluents and incubating them at 65 ºC overnight. The DNAs were recovered by proteinase K and RNase A digestion, followed by phenol/chloroform extraction and ethanol precipitation. Pellets were resuspended in 50 μl of 10 mM Tris-HCl [pH 8.0]. ChIP-DNA was quantified using the Qubit dsDNA High Sensitivity Assay kit (Thermo Fisher Scientific). Libraries were prepared according to Nugen's instructions accompanying their Ovation Ultralow System V2 kit.