GSM7108309: Histone_3_K27_acetylation, DIPG4, DMSO, radiation, replicate 1; Homo sapiens; ChIP-Seq
Cells were crosslinked with 1% formaldehyde added to the growth media for 10 min, followed by quenching with 0.125M glycine for 5 min. Cells were washed twice with cold PBS, collected in ice-cold PBS by scraping, pelleted, and resuspended in cell lysis buffer (5mM PIPES, pH 8.0; 85mM KCl; 0.5% NP-40). Following incubation on ice for 10 min, a nuclear-enriched fraction was collected by centrifugation for 5 min at 2500 xg at 4 C. Pellet was resuspended in ChIP lysis buffer (50mM Tris-HCl, pH 8.0; 10mM EDTA; 1% SDS) containing COmplete EDTA free protease inhibitors (Sigma) and quantified with BCA. 2ug/ul of lysates were sonicated with a Bioruptor Plus (Diagenode) for 20 cycles (30 seconds ON and 30 seconds OFF). The size of the sonicated DNA fragments was checked on an 1X TAE 1% agarose gel electrophoresis and ranged between 250 to 500 bp. After removing the debris via centrifugation, chromatin extracts were collected.   For immunoprecipitation, chromatin extracts were diluted with Chip Dilution Buffer (16.7mM Tris-HCl, pH8.1, 1.2Mm EDTA, 167mM NaCl, 0.01%SDS and 1.1% Triton x100) incubated with primary antibodies anti-K27Ac (#39133, Active Motif) overnight at 4 C.  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 4 C. 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 cross-links were reversed by adding NaCl (a final concentration 0.2 M) into the eluents and incubating them at 65  C overnight. The DNA was recovered by proteinase K and RNase A digestion, followed by phenol/chloroform extraction and ethanol precipitation. Pellets were resuspended in 25 ul of RNase and DNase free water, and ChIP-DNA was quantified using the Qubit dsDNA High Sensitivity Assay kit (Thermo Fisher Scientific), followed by library preparation. The NEBNext Ultra DNA Library Prep Kit for Illumina (NEB #E7645S) with Dual Index Primers (NEB #E7600) were used to library preparation for ChIP-seq.    NEBNext End Prep  1x TE was added to the DNA to get final volume of 50 ul and mixed with 3 ul of NEBNext Ultra II End Prep Enzyme Mix and 7 ul of NEBNext Ultra II End Prep Reaction Buffer. This was placed in a thermocycler with heated lid set to 75 C and run for 30 minutes at 20 C, 60 minutes at 50 C, then held at 4 C.  Adaptor Ligation  The adaptor ligation reaction was performed with 60 ul of Prep Reaction Mixture, 2.5 ul of NEBNext Adaptor for Illumina diluted following the manufacture recommendations, 30 ul of NEBNext Ultra Ligation Master Mix, and 1 ul of NEBNext Ligation Enhancer, incubated at 20 C for 15 min with heated lid off. 3 ul of USER Enzyme was then added to the reaction and incubated at 37 C for 15 min with heated led set to 47 C.  Cleanup of Adaptor-Ligated DNA without size selection  1.1x of AMPure XP Beads were added to the Adaptor Ligation reaction, and samples were incubated for 5 min at room temperature. Tubes were placed on a magnetic stand for 5 min to separate beads from supernatant, then supernatant was removed and beads were washed 2 times with 200 ul of 80% freshly prepared ethanol. Beads were air dried for up 5 min while tubes were on magnet with lid open. DNA was eluted from the beads by adding 17 ul of 0.1XTE buffer.   PCR Enrichment of Adaptor-ligated DNA  To 15 ul of the Adaptor Ligated DNA fragments was added 25 ul of NEBNext Ultra Q5 Master Mix, 5 ul of Index primer i7 and 5 ul Universal PCR Primer i5, and PCR amplification was performed following the cycling conditions: 98 C for 45s, 98 C 15s and 60 C 10s for 16 cycles, 72 C for 1 min, and hold at 4 C. Followed by a clean up of the PCR reaction using AMPure XP Beads.   The DNA was quantified using Qubit, and the quality and size of libraries were checked at the Bioanalyzer prior to sequencing.