title: GSM2486297: SF7761 H3.3K27M ChIP-seq repeat 1; Homo sapiens; ChIP-Seq
library_construction_protocol: Frozen tissues were cut, divided into 50 mg aliquots, and stored in tubes at -80°C. The frozen tissues were homogenized on ice for 15–30 seconds in 500 μL 1X PBS using a tissue grinder (ACTGene). Tissue homogenates or cultured cells were cross-linked with 1% formaldehyde (final concentration) for 10 min and quenched with 125 mM glycine for 5 min at room temperature and washed once with TBS. The pellets were resuspended in cell lysis buffer (10 mM Tris-HCl, pH7.5, 10 mM NaCl, 0.5% NP-40) and incubated on ice for 10 min. Lysates were divided into two aliquots and washed with MNase digestion buffer (20 mM Tris-HCl, pH7.5, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2). After re-suspending in 500 μL MNase digestion buffer containing a proteinase inhibitor cocktail (Sigma), the lysates were incubated in the presence of 1,000 units of MNase (NEB, Ipswich, MA, Cat.# M0247S) at 37 °C for 20 min with continuous mixing in thermal mixer (Fisher Scientific, Pittsburgh, PA). After adding the same volume of sonication buffer (100 mM Tris-HCl, pH8.1, 20 mM EDTA, 200 mM NaCl, 2% Triton X-100, 0.2% sodium deoxycholate), the lysates were sonicated for 15 min (30 secs on / 30 secs off) using Bioruptor Twin (UCD-400) (Diagenode) and centrifuged at 21,130 x g for 10 min. The supernants were collected and the chromatin content was estimated by the Qubit assay (Invitrogen). For the normalization of ChIP efficiency, S2 or Sf9 chromatin was added. The chromatin was then incubated with 5 µg of rabbit monoclonal anti-Ezh2 antibody (Cell Signaling, Cat.# 5246), 5 µg of rabbit monoclonal anti-H3K27me3 antibody (Cell Signaling, Cat.# 9733), 2 µg of rabbit polyclonal anti-H3K27M antibody (Millipore, Cat.# abe419), or 5 µg of mouse monoclonal anti-Jarid2 antibody (Novus, Cat.# NB100-2214) on a rocker overnight. Protein G-magnetic beads (30 μl, Life Technologies) were added for 3 hours incubation. The beads were extensively washed with ChIP buffer (50 mM Tris-HCl, pH8.1, 10 mM EDTA, 100 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate), high salt buffer (50 mM Tris-HCl, pH8.1, 10 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate), LiCl buffer (10 mM Tris-HCl, pH8.0, 0.25 M LiCl2, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA), and TE buffer. Bound chromatin was eluted and reverse-crosslinked at 65°C overnight. DNAs were purified using Min-Elute PCR purification kit (Qiagen) after the treatment of RNase A and proteinase K. ChIP-seq libraries were prepared from 10 ng ChIP and input DNA using the Ovation ultralow DR Multiplex kit (NuGEN). The ChIP-seq libraries were sequenced to 51 base pairs from both ends on an Illumina HiSeq 2000 instrument in the Mayo Clinic Center for Individualized Medicine Medical Genomics Facility.
attributes.tag: GEO Accession
attributes.value: GSM2486297