ChIP-Atlas: SRX655399

Antigen

source_name: Pediatric, K27M mutated DIPG cells treated with GSKJ4 for 24h, H3K27me3 ChIP
cell line: SF8628
age: year 3
tissue: pons
gender: female
treatment: GSKJ4
treatment duration: 24h
chip antibody: anti-H3K27me3

library_strategy: ChIP-Seq
library_source: GENOMIC
library_selection: ChIP
library_construction_protocol: Briefly, 1×10^6 cells, treated with vehicle or 6 μM GSKJ4 for 24 and 72 hours, were harvested, washed with PBS and fixed with 1% formaldehyde for 5 minutes at room temperature. Cells were then quenched with 125 mM glycine for 5 minutes. After wash, cell extracts were prepared using lysis buffer (50 mM HEPES/KOH at pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton-X100, 0.1% Na-deoxycholate, 1 mM PMSF, 1 mM Pefoblock, 1 mM benzamidine, 1 mg/ml bacitracin) on ice for 10 minutes. Chromatin was sheared by sonication (bioruptor, High power, 15 × 2 cycles), to average lengths of 500 bp, then immunoprecipitated using an antibody against histone H3K27me3 (#9733, Cell Signaling). After repeated washings, the DNA was recovered from the beads by incubating the beads in elution buffer (10 mM Tris at pH 8.0, 10 mM EDTA at pH 8.0, 1% SDS, 150 mM NaCl, 5 mM DTT) at 65 oC. Both input DNA and eluted DNA were subsequently purified using Qiagen Mini Elute PCR purification kit. ChIP DNA libraries were prepared with the Ovation Ultralow DR Multiplex system (NuGEN).