title: GSM2676331: H3K9me3_TAM00hr_rep1; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were treated with ethanol or tamoxifen (1 µM) for 24 hours and then cross-linked using ethylene glycol bis (succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG) and disuccinimidyl suberate (DSS) mixture (2.5 µM each) for 45 minutes at room temperature. After this initial crosslinking, cells were further fixed using 1% formaldehyde for 20 minutes at room temperature and then quenched by glycine (0.125 M). Chromatin in sonication buffer (50 mM HEPES, pH7.5, 140 mM NaCl, 2mM EDTA, 2mM EGTA, 1% Triton-100 and 0.4% SDS) was sheared using Branson Microtip Sonifier 450 (12 cycles of 15 second at a sonication setting of output 4 and duty cycle 60%) to a size mostly between 100-150 bp.  The sonicated chromatin solution was diluted to 0.085% SDS and immunoprecipitated with antibodies against H3K4me3 (ab8580), H3K27ac (ab4729), H3K4me1 (ab8895), H3K36me3 (ab9050), H3K27me3 (ab6002) and H3K9me3 (ab8898).  Immunoprecipitated chromatin was decrosslinked using RNase Cocktail (Ambion, AM2286) and Pronase (Roche, 10165921001). ChIP DNA was end repaired, addition of “A” and adapters ligation and PCR amplification to produce ChIP-seq libraries. The DNA concentration was measured by Bioanalyzer before sequencing using Hiseq 2000 at the Bauer Core Facility, Harvard.
attributes.tag: GEO Accession
attributes.value: GSM2676331