GSM4793272: H3K27M_H3K27me3_rep2; Homo sapiens; ChIP-Seq
The cell pellets were resuspended in 3 ml cell lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% IPGAL including protease inhibitors) and incubated on ice for 10 minutes, spun at 5000 rpm for 5 min, and the supernatant was aspirated. Nuclei were resuspended in 1ml nuclear lysis buffer (50 mM Tris.HCl pH8.0, 10 mM EDTA, 1% SDS plus protease inhibitors) and incubated on ice for 10 minutes' cold room rocker. Chromatin was then sonicated with a Biorupter (diagenode) 30 seconds on/off, repeated for 12 cycles total. After sonication, thcells were centrifuged at maximum rpm at RT for 3 minutes, and the supernatant was collected in a new tube. The sheared chromatin was confirmed to be between 300 - 500 bp by agarose gel electrophoresis, and the samples were stored at -80C until used. The 100 µg of chromatin was diluted in ChIP dilution buffer (16.7 mM Tris–HCl (pH 8.1), 167 mM NaCl, 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA)was incubated overnight at 4°C with primary antibodies H3K27me3 (#07-449, Millipore Sigma), and IgG (#PP64-10-KC, Millipore Sigma) in the cold room. The next day, Protein A Dynabeads (Invitrogen) was washed three times in ChIP dilution buffer and added to the IP chromatin, and then the incubation was further continued for 4 hours. After a 4-h incubation, the beads were washed sequentially with 1× low-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris (pH 8), 150 mM NaCl], 1× high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris (pH 8), 500 mM NaCl), 2× LiCl buffer (0.25 M LiCl, 1% IGEPAL CA- 630 (Sigma), 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris, pH 8.0]. The beads pellet was resuspended in 125 µl of elution buffer (1% SDS, 5 mM DTT, and 150 mM NaCl in Tris-EDTA buffer) and incubated at 65°C for 10 minutes. The supernatant was collected in a new tube, and the elution was repeated again. The DNA was reverse cross-linked (with 200mM NaCl, 1% SDS, 5 mM DTT) at 65°C overnight and 250 µg Protenease K was added and incubated at 37°C for 1 hour. The DNA samples were purified using the NucleoSpin gel/PCR purification kit (Takara). DNA quantification was performed using the Qubit 2.0 fluorometer (Invitrogen), and DNA quality was further confirmed using an Agilent Bioanalyzer 2100 system and high-sensitivity DNA chips. For each library preparation, 1.5 ng of immunoprecipitated chromatin was used for Illumina sequencing-compatible library preparation with the DNA Smart ChIP-seq kit (Takara), as recommended in the manufacturer's protocol. To assess ChIP enrichment by qPCR, 200–300 pg ChIP and input DNA were used as starting material for amplicon amplification with the 7500 Fast Real-Time PCR System (Applied Biosystems). Enrichment was calculated from at least four biological replicates, with four technical replicates per biological replicate performed. DNA sizing was determined using an Agilent Bioanalyzer, with the concentration determined by Qubit assay.  Library preparation was performed with up to 10ng of DNA. DNA was blunt ended, had an A base added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends. Size selection was performed with Ampure XP beads (Beckman Coulter) to select for fragments between 150-700bp. Ligated fragments were then amplified for 15 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq-6000 using paired end reads extending 150 bases.