Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Histone
Antigen
H3K9K14ac

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
strain
129/Ola
genotype
Wildtype (E14Tg2a)
treatment
Control
chip antibody
H3K9,K14ac (Millipore, Catalog# 06-599, Lot# 2019589)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ES cells were crosslinked with 1% formaldehyde at room temperature for 10 min, followed by 5 min incubation with 125mM glycine at room temperature. Cells were washed in PBS, flash frozen in liquid nitrogen and stored at –80°C prior to use. Cell pellets were thawed on ice, resuspended in lysis buffer 1 (50mM Hepes KOH, pH 7.9, 140mM NaCl, 1mM EDTA , 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100) and incubated for 10 min on a rotator at 4°C. Cells were washed in TE (10mM Tris-HCl, pH 8, 1mM EDTA). Cells were resuspended in TE containing 0.5% SDS, incubated on ice for 10 min and sonicated using a Bioruptor (Diagenode) on High setting for 18 cycles of 30 sec (30 sec pause between pulses). The sonicated extract was centrifuged at 20000g for 10 min at 4°C and the supernatant diluted 5 fold with ChIP dilution buffer (20mM Tris-HCl, pH 8, 150mM NaCl, 1% Triton X-100, 1mM EDTA). ChIP DNA was end repaired by incubation at 20°C for 30 min with 3 U of T4 DNA Polymerase (NEB), 10 U of Polynucleotide Kinase (NEB), 2 U DNA Polymerase I Large (Klenow) fragment (NEB), 1x T4 DNA ligase reaction buffer (NEB) and 400 nM dNTPs. The enzymes were then heat inactivated at 75ºC for 20 min, after which the DNA was ethanol precipitated. A tail of 'A' bases was added to the 3’ ends of the DNA by incubation with 5 U Klenow Fragment (3’-5’ exo-; NEB), 200nM dATP and 1x buffer 2 (NEB) at 37°C for 30 min. The enzymes were heat inactivated and cleaned up as before .Barcoded Ilumina paired end adaptors were then ligated to the processed ChIP DNA by incubation with 300 U of T4 DNA ligase (NEB), 1x T4 DNA ligase buffer (NEB), 7.5% PEG-6000 and 2 pmol of annealed Illumina adaptors for 3 hr at room temperature. Ligated DNA was purified using MinElute PCR columns (Qiagen) and eluted in 10 μl water. Ligated DNA was amplified by 16-18 cycles of PCR with primers complementary to the adaptor sequences and Phusion 2x premix (Finnzymes). The DNA was purified using QIAquick PCR Purification columns (Qiagen).

Sequencing Platform

instrument_model
Illumina HiSeq 2000

mm10

Number of total reads
18272446
Reads aligned (%)
98.0
Duplicates removed (%)
12.2
Number of peaks
12284 (qval < 1E-05)

mm9

Number of total reads
18272446
Reads aligned (%)
97.9
Duplicates removed (%)
12.5
Number of peaks
12271 (qval < 1E-05)

Base call quality data from DBCLS SRA