GSM6454196: H3K27Ac_histone H3_acetyl K27_DIPG4_with_DSMO_Radiation 2; Homo sapiens; ChIP-Seq
Cells were crosslinked with 1% formaldehyde added to the growth media for 10min, followed by quenching with 0.125M glycine for 5min. Cells were washed twice with cold PBS, collected in ice-cold PBS by scrapping, 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 5min at 2500 xg at 4oC. 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 TAE1 % 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 4ºC.  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 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 25ul 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 ChiPseq and Cut and Run samples sequencing.    NEBNext End Prep  1x TE was added to the DNA to get final volume of 50ul and mixed with 3ul of NEBNext Ultra II End Prep Enzyme Mix and 7ul of NEBNext Ultra II End Prep Reaction Buffer, placed in thermocycler with heated lid set to 75oC and run the program 30 minutes at 20oC, 60 minutes at 50oC and hold at 4oC.  Adaptor Ligation  The adaptor ligation reaction was performed with 60ul of Prep Reaction Mixture, 2.5ul of NEBNext Adaptor for Illumina diluted following the manufacture recommendations, 30ul of NEBNext Ultra Ligation Master Mix and 1ul of NEBNext Ligation Enhancer, incubated at 20oC for 15min with heated lid off. 3ul of USER Enzyme was added to the reaction and incubated at 37oC for 15min with heated led set to 47oC.  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 5min at room temperature. Tubes were placed on a magnetic stand to separate beads from supernatant, after 5 min, supernatant was removed, and beads were washed 2 times with 200ul of 80% freshy 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 17ul of 0.1XTE buffer.   PCR Enrichment of Adaptor-ligated DNA  To 15ul of the Adaptor Ligated DNA fragments was added 25ul of NEBNext Ultra Q5 Master Mix, 5ul of Index primer i7 and 5ul Universal PCR Primer i5, and PCR amplification was performed following the cycling conditions: 98oC for 45s, 98oC 15s and 60oC 10s for 16 cycles, 72oC for 1min and hold at 4oC. 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 and sent for sequencing at Novogene Corporation Inc.