title: GSM1621017: PMLKO_H33; Mus musculus; ChIP-Seq
library_construction_protocol: For ChIP-seq: Cells (10e7 cells/ChIP) were harvested and fixed with 1% formaldehyde for 10 min before quenching with 125 mM glycine. Cells were lysed for 30 min at 4C on a rotator in RIPA buffer (140 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% deoxycholate, 1 mM PMSF, protease inhibitor cocktail) adjusted to 1% SDS, and sonicated 3 times 15 min in a Bioruptor® (Diagenode). After sedimentation at 10,000 g for 10 min, the supernatant was collected and diluted 10x in RIPA buffer without SDS to constitute input chromatin. Chromatin was incubated overnight at 4oC on a rotator with antibodies coupled to magnetic Dynabeads Protein G. ChIP samples were washed 3 times in ice-cold RIPA buffer. Crosslinks were reversed and DNA eluted for 6 h at 37C in 50 mM NaCl, 20 mM Tris-HCl pH 7.5, 5 mM EDTA, 1% SDS, 0.5 µg/ml RNase A, and 2 µg/ml Proteinase K added twice. DNA was purified and dissolved in H2O.For RNA-seq: 10e6 cells were cultured. For RNA-seq: Total RNA was isolated using the Ambion TRIzol® Reagent RNA extraction kit (Life Technologies), quality checked using a BioAnalyzer 2100 and processed for library preparation. Sequencing library was prepared according to the Illumina protocol, and sequenced on an Illumina HiSeq2500 at the Norwegian Sequencing Center.
attributes.tag: GEO Accession
attributes.value: GSM1621017