Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Input control
Antigen
Input control

Cell type

Cell type Class
Pluripotent stem cell
Cell type
mESC derived mesodermal cells
NA
NA

Attributes by original data submitter

Sample

source_name
MES stage cultures, Whole cell extract
strain
129/Ola
time point
Differentiation Day 4
cell type
mesodermal cells (MES)
antibody
None (Whole cell extract)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Chromatin immunoprecipitation of histone modifications were performed according to the Young lab protocol (Lee et al., 2006) with minor modification. Briefly, frozen pellets of cross-linked cells (10 3 106) were thawed in cold lysis buffer 1 (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1 3 protease inhibitors) and gently rocked at 4 C for 10 min in 14 ml conical tubes. Cells were pelleted at 1350 x g at 4 C in a clinical centrifuge and resuspended in cold lysis buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1 3 protease inhibitors) and gently rocked at 4 C for 10 min in 14 ml conical tubes. Cells were pelleted at 1350 x g at 4C in a table top centrifuge and resuspended in 2 ml cold lysis buffer 3 (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine, 1 3 protease inhibitors) and sonicated to 200-600 bp fragments using a Diagenode Bioruptor (3 3 10 min cycles, 30 s ON / 30 s OFF at 4 C). Soni- cated lysates were cleared by pelleting insoluble material at 20,000 x g at 4 C followed by incubation with antibody bound Protein A/G magnetic beads (2.5 mg Ab / 50uL beads / IP) in 1 ml of 0.5% BSA/PBS overnight at 4 C. Magnetic beads were washed 3 times with block (0.5% BSA/PBS), incubated for approximately 4 hr at 4 C with antibody in block and then washed 3 times with block prior to addition of cleared cell lysates. Immunoprecipitated material was washed five times with cold wash buffer (RIPA: 50 mM HEPES-KOH, pKa 7.55, 500 mM LiCl, 1 mM EDTA, 1.0% NP-40, 0.7% Na-Deoxycholate) and one time with TE plus NaCl, followed by elution and uncrosslinking in 210 uL of 1% SDS in TE overnight at 65 C. 200 uL of uncrosslinked material was treated with RNase A for 2 hr, proteinase K for 2 hr and extracted 2 times with phenol chloroform isoamyl alcohol, followed by ethanol precipitation with a glycogen coprecipitant, 80% ethanol wash and final resuspension in TE. Illumina sequencing libraries were generated (Schmidt et al., 2009), with minor modi- fication. Briefly, 5-50 ng of immunoprecipitated nucleic acid was end repaired, a-tailed and ligated to Illumina single end adapters using an NEB Next kit (New England Biolabs). 200-300 bp adaptor ligated nucleic acid was gel purified and enriched via 19 cycles of PCR amplification with Phusion High-Fidelity DNA Polymerase, followed by sequencing on an Illumina HiSeq 2000 system

Sequencing Platform

instrument_model
Illumina HiSeq 2000

mm10

Number of total reads
114482019
Reads aligned (%)
97.4
Duplicates removed (%)
21.5
Number of peaks
889 (qval < 1E-05)

mm9

Number of total reads
114482019
Reads aligned (%)
97.2
Duplicates removed (%)
21.5
Number of peaks
1025 (qval < 1E-05)

Base call quality data from DBCLS SRA