alias: GSM3831300
title: GSM3831300: K27M-SU-DIPGXIII_WCE; Homo sapiens; ChIP-Seq
library_construction_protocol: For H3K27ac, H3K27me3, and H3K4me1 chIP-seq, nuclei were using a hypotonic buffer (25 mM Tris (pH 8.0), 1.5 mM MgCl2, 0.2% NP40, 10 mM KCl). Isolated nuclei were washed in buffer A (0.34 M sucrose, 4 mM MgCl2, 60 mM KCl, 50 mM HEPES (pH 7.4), and digested with micrococcal nuclease I (New England Biotechnology) to obtain mono- and di-nucleosomes. Micrococcal nuclease reactions were stopped by the addition of EGTA to 10 mM, nuclei were then pelleted and chromatin was extracted in 10 mM EDTA. Buffer B (20 mM Tris (pH 8.0), 5 mM EDTA (pH 8), 500 mM NaCl, ) was added to dilute each sample to 100 ng/µl before incubating overnight at 4°C with 5 µg of the following antibodies: H3K27ac (Active Motif, #39135), H3K4me1 (Abcam, ab8895), or H3K27me3 (Millipore, 07-449). Antibody-chromatin complexes were retrieved using Protein G Magnetic Beads (EMD Millipore), followed by extensive washing and elution  in a buffer containing 1% SDS, 50 mM Tris HCl (pH 7.0), and 1 mM EDTA. For H3K27M ChIP-seq 10 million SU-DIPGXIII cells were crosslinked with 1% formaldehyde added directly to the media for 10 minutes at 37°C before quenching with glycine added to acheive a final concentration of 0.11 mM. After pelleting and washing cells in twice with PBS, cells were re-suspended in lysis buffer (50 mM Tris-HCl, 1% SDS, 0.25% sodium deoxycholate) with protease inhibitors added and lysed for 10 minutes on ice. After lysis samples were diluted 1:2.33 with ChIP dilution buffer (50 mM Tris-HCl pH 7.4, 0.01% SDS, 15 0mM NaCl, and 1% Triton-X100)  and then sonicated to 200-3000 base pair fragments. After sonication samples were diluted another 10 fold  in ChIP dilution buffer and H3K27M immunoprecipitation was done overnight using 10 µg of antibody (Millipore, ABE419) followed by retrieval of immunoprecipitated proteins and DNA with protein G beads on a rotator at 4°C for 2 hours. Protein G beads were then washed with 1 mL RIPA wash buffer (0.1% SDS, 0.1% sodium deoxycholate 1% Triton X100, 1 mM EDTA, 10 mM Tris-HCl pH 8.1, 150 mM NaCl), followed by 1 mL RIPA wash buffer with the NaCl concentration increased to 500 mM, and 1 mL LiCl wash buffer (10 mM Tris-HCl, pH 8.1, 250 mM LiCl, 0.5% TritonX-100, 0.5% sodium deoxycholate), and 1 mL of 10 mM Tris-HCl (pH 8.5). ChIP samples were eluted with a solution of 50 mM Tris-HCl (pH 8.0), 0.1% SDS, 150 mM NaCl and 5 mM dithiotreitol (DTT) at 65º C for 1 hour. Eluates were then subjected to treatment RNAse (Roche # 11119915001) at 37°C for 30 minutes followed by treatment with Proteinase K (Sigma# G1508) added to 0.25 mg/mL at 63°C for 3 hours. DNA was then isolated by double sided SPRI bead purification using AMPure XP beads following the manufacturer's protocol. Libraries for sequencing were prepared using the NEBNext library preparation kit (New England Biotechnology) and sequenced on an Illumina HiSeq2500 to a length of 50 base pairs except in the case of H3K27M chIP libraries, which were analyzed by paired end sequencing to a length of 40 base pairs.
identifiers.namespace: GEO
identifiers.id: GSM3831300