Sample information curated by ChIP-Atlas

Antigen

Antigen Class
RNA polymerase
Antigen
RNA polymerase II

Cell type

Cell type Class
Pluripotent stem cell
Cell type
ES cells
NA
NA

Attributes by original data submitter

Sample

source_name
WT_4H8_mESCs
strain background
129
genotype/variation
WT
cell type
mESCs
chip antibody
anti-RNA pol II (Millipore, #05-623)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Prior to chromatin immunoprecipitation, antibodies (5 μl polyclonal or 5 μg monoclonal) were pre-bound to 200 μl Protein G magnetic beads (Invitrogen, #10004D) by overnight incubation in PBS with 5% BSA; beads were washed and resuspended in PBS with 5% BSA. MEF-depleted mESC were fixed at room temperature with 1% formaldehyde (Sigma-Aldrich, # 252549) for 5 min; fixation was stopped by adding glycine (ICN Biomedical, #ICN808822) to 125 mM. Cell membranes were lysed with cold NP-40 buffer (1% NP40, 150 mM NaCl, 50 mM Tris–HCl; pH 8.0) and nuclei collected by centrifugation at 12,000 × g for 1 min at 4 °C. Nuclear pellets were resuspended at a concentration of 2E8 cells/ml in ChIP sonication buffer (1% SDS, 10 mM EDTA, 50 mM Tris–HCl; pH 8.0) supplemented with protease (Thermo Scientific, #78430) and phosphatase (Calbiochem, #524627) inhibitor cocktails. Chromatin was sheared into 150-400 bp fragments by sonication (Bioruptor Twin, Diagenode). Debris was pelleted by centrifugation and cleared chromatin was diluted 10-fold in ChIP dilution buffer (1.1% Triton X-100, 0.01% SDS, 167 mM NaCl, 1.2 mM EDTA, 16.7 mM Tris–HCl; pH 8.1). The prepared antibody-bound beads were added to 1 ml diluted chromatin containing 2E7 million cell equivalents and incubated overnight with rotation at 4 °C. Immune complexes were washed 5 times with LiCl wash buffer (250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 100 mM Tris-HCl; pH 7.5) and once with TE (0.1 mM EDTA, 10 mM Tris-HCl; 7.5). Beads were resuspended in IP Elution Buffer (1% SDS, 0.1 M NaHCO3) and crosslinks reversed by overnight incubation at 65 °C. DNA was purified by column purification (QIAGEN, # 28106) Sequencing libraries were constructed from DNA samples with the Illumina TruSeq V3 library construction protocol.

Sequencing Platform

instrument_model
NextSeq 550

mm10

Number of total reads
57963889
Reads aligned (%)
98.0
Duplicates removed (%)
10.7
Number of peaks
725 (qval < 1E-05)

mm9

Number of total reads
57963889
Reads aligned (%)
97.8
Duplicates removed (%)
10.7
Number of peaks
754 (qval < 1E-05)

Base call quality data from DBCLS SRA