GSM1382268: ES DNMT3B1 S277P rep2; Mus musculus; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Unclassified
Antigen
Unclassified
Cell type
Cell type Class
Pluripotent stem cell
Cell type
ES cells
NA
NA
Attributes by original data submitter
Sample
source_name
embryonic stem cells, mock treatment
cell type
embryonic stem cells
genotype
wild type
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
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, 0.1% SDS and 150 mM NaCl for 2 hours on ice. Crosslinked chromatin was subjected to sonication in a Bioruptor instrument (Diagenode). 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 Illumina TruSeq library protocol with index adapters. Four to five libraries with different barcodes were pooled at equimolar ratios per lane