Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Input control
Antigen
Input control

Cell type

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

Attributes by original data submitter

Sample

source_name
E14Tg2a mESCs
gender
male
strain
129P2/OlaHsd
cell type
Embryonic Stem Cells
antibody
none

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Trypsinised ESCs (20x106) were washed twice in PBS. Cells were resuspended in 250mls of PBS and fixed by the addition of an equal volume of 2% methanol-free formaldehyde (Thermo Scientific Pierce PN28906; final concentration of 1%) and incubated at RT for 10 mins. Fixation was stopped by 5 min incubation with 125mM glycine at room temperature. Cells were washed in PBS prior to lysis. All of the buffers were supplemented with the following additives just prior to use (0.2 mM PMSF, 1 mM DTT, 1x Protease inhibitors (Calbiochem, 539134-1SET)). Cell pellets were resuspended in lysis buffer 1 (50mM Tris-HCl pH 8.1, 10mM EDTA and 20% SDS) and incubated for 10 min at 4°C. Lysates were diluted 1:10 in ChIP dilution buffer (0.1% Triton X-100, 2mM EDTA, 150mM NaCl, 20mM and Tris-HCl pH8.1) and sonicated using a chilled Bioruptor (Diagenode; 40x 1 min cycles of 30sec on / 30sec off on ‘high’ setting at 4°C). The sonicated extract was pre-cleared by centrifugation at 16000g for 10 min at 4°C. The supernatant was transferred to a fresh tube and supplemented with BSA and triton X-100 to a final concentration of 25mg/ml and 1% respectively. A sample of the chromatin was retained as an input reference. Antibodies were pre-coupled to a 1:1 mix of protein A and G Dynabeads (Life Technologies; 10001D and 10004D respectively) at a ratio of 1mg antibody per 30ml of dynabeads. 12x106 and 6x106 cell equivalents of lysate were added to 10ug of Anti-Ring1B (MBL D139-3) or 5ug anti-H3K27me3 (Millipore 07-449) respectively and incubated for 10hrs on a rotating wheel at 4ºC. Bead-associated immune complexes were washed sequentially with wash buffers A, B and C for 10 mins at 4ºC on a rotating wheel followed by 2 washes in TE buffer at RT (wash buffer A - 1% Triton X-100, 2mM EDTA, 150mM NaCl, 20mM and Tris-HCl pH8.1; wash buffer B - 1% Triton X-100, 0.1% Sodium-Deoxycolate, 0.1% SDS, 1mM EDTA, 500mM NaCl, 20mM and Tris-HCl pH8.1; wash buffer C – 1% NP40, 0.1% Sodium-Deoxycolate, 1mM EDTA, 250mM LiCl, 20mM and Tris-HCl pH8.1). Chromatin was released from the beads by incubation with elution buffer (0.1 M NaHCO3 and 1 % SDS) for 15 mins at 37 ºC followed by the addition of RNaseA and Tris pH6.8 (final concentration of 20mg/ml and 100mM respectively) and incubation at 65ºC for 2 hours following by the addition of 50mg of proteinase K and incubation at 65ºC overnight to degrade proteins and reverse the cross-links. Dynabeads were removed using a magnetic rack and the chromatin purified using PCR Purification columns (Qiagen) according to manufacturer’s instructions. libraries were prepared as previously described (Bowman et al., 2013 - PMID:23837789) with the following modifications. No purification was performed between the A-tailing reaction and the ligation reactions; instead the ligation was supplemented with ligation reagents (400 U of T4 DNA ligase (NEB), 1x buffer 2 (NEB), 7.5% PEG-6000, 1mM ATP and 13.3nM of annealed Illumina adaptors (AU) and incubated at 25°C for 90min. Size selection purifications following the ligation and amplification PCR steps were performed with 1x and 0.8x reaction volumes of Agencourt AMPure XP beads (Beckman Coulter - A63880). Six samples were multiplexed per Hi-seq lane.

Sequencing Platform

instrument_model
Illumina HiSeq 2000

mm10

Number of total reads
38799682
Reads aligned (%)
97.8
Duplicates removed (%)
17.7
Number of peaks
722 (qval < 1E-05)

mm9

Number of total reads
38799682
Reads aligned (%)
97.5
Duplicates removed (%)
17.7
Number of peaks
795 (qval < 1E-05)

Base call quality data from DBCLS SRA