GSM3597739: Transformed_H3K27me3_rep2; Homo sapiens; ChIP-Seq
Cells were fixed with 1% formaldehyde in cell culture media for 10 minutes at room temperature. Cells were lysed in 1:1 SDS-containing buffer (100mM NaCl, 50mM Tris-HCl pH 8.0, 5mM EDTA pH 8.0, 0.2% NaN3, 0.5% SDS): triton-containing dilution buffer (100mM NaCl, 500mM Tris-HCl pH 8.6, 5mM EDTA pH 8.0, 0.2% NaN3, 5% Triton X-100) plus protease inhibitors (Cell Signalling). Chromatin was then sheared to 200-400bp using a Biouptor Pico Sonicator (Diagenode). Co-immunoprecipitation was performed by combining 1mg of clarified chromatin lysate and EZH2 (Cell Signalling - 5246S, 1:40) or H3K27me3 (Upstate - 07-449, 1:100) antibodies, which were then rotated at 4⁰C o/n. The following day, protein-G magnetic beads (ThermoFisher) were added to the immunoprecipitations and rotated at 4⁰C for 4 hours. The bead-antibody complexes were washed and chromatin was eluted by shaking at 65⁰C o/n 0.1M NaHCO3 1% SDS solution. Chromatin was isolated by PCR purification (Qiagen). Library preparation was undertaken with 5-10ng of DNA using the Illumina TruSeq ChIP protocol.
GEO Accession
GSM3597739