title: GSM2824373: ES_bioDNMT3A1_rep2; Mus musculus; ChIP-Seq
library_construction_protocol: For cross-linking and chromatin extraction, cells were fixed for 10 minutes with 1% Formaldehyde at room temperature and incubated for 10 min on ice in presence of 1.2M Glycine. Cells were harvested and treated for 10 min with 10mM EDTA, 10mM TRIS, 0.5mM EGTA and 0.25% Triton X-100 and 10 min in 1mM EDTA, 10mM TRIS, 0.5mM EGTA and 200mM NaCl with subsequent lysis in 50mM HEPES, 1mM EDTA, 1% Triton X-100, 0.1% deoxycholate, 0.2% SDS and 300 mM NaCl for 2 hours on ice. Crosslinked chromatin was subjected to sonication in a Bioruptor Pico instrument (Diagenode) and diluted with equal volumes of 50mM HEPES, 1mM EDTA, 1% Triton X-100, 0.1% deoxycholate after successful sonication. ProteinA-dynabeads pre-cleared 150-250 µg chromatin were incubated with 40 µl blocked (1%CFSG, 100ng tRNA) Streptavidin-M280 magnetic beads over night at 4°C. Beads were washed with two rounds of 2% SDS, 1x high salt buffer, 1x LiCl Buffer and two rounds of TE. Beads were treated with RNaseA for 30 min at 37°C, Proteinase K for 3 hours at 50°C then de-crosslinked over night at 55°C. DNA was purified with Phenol-Chloroform extraction and EtOH precipitation. ChIP-seq library using NEB ChIPlibrary protocol with index adapters.
attributes.tag: GEO Accession
attributes.value: GSM2824373