Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Histone
Antigen
H3K27ac

Cell type

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

Attributes by original data submitter

Sample

source_name
MC1-ZE7 ES cells
cell type
FACS sorted Emerald+ ES cells
strain
MC1 (129S6/SvEvTac)
chip antibody
H3K27ac (Active Motif #39133)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
FACS sorted MC1-ZE7 cells were cross-linked with 1% formaldehyde for 10 min at room temperature. The reaction was stopped by 125 mM glycin. The cells were washed with PBS and stored at –80°C prior to use. The cells were lysed in Lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS) containing proteinase inhibitor cocktail (Roche). Sonication was coducted with the Misonix sonicator XL to generate DNA fragments of approximately 150-450 bp. The sonicated lysates were diluted in ChIP dilution buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100) containing proteinase inhibitor cocktail and incubated overnight at 4°C with 30 µl of protein G magnetic beads (Invitrogen) that had been preincubated for 3 h with 3 µg of antibodies. The precipitants were washed three times with RIPA buffer (10 mM Tris-HCl, pH 7.5, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate) and once with 10 mM Tris-HCl, pH 8.0, 5 mM EDTA, 10 mM NaCl. Bound chromatin was eluted from the beads at 70°C, 1,300 r.p.m in Elution buffer (20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 50 mM NaCl, 1% SDS), followed by crosslink reversal at 70°C for 6 h. After treatment with RNase A and proteinase K, DNA was purified by a phenol-chloroform-isoamylalcohol extraction and isopropanol precipitation. Library preparation was performed using ChIP-seq sample preparation kit (Illumina) with several modifications as follows. A 1:30 dilution of the Adaptor Oligo Mix was used in the ligation step. A range of fragment size (200-300 bp) was selected by separation on a 2% low melting temperature agarose gel (Lonza). DNA purification in each reaction was carried out using AMPure XP beads (Beckman). In the experiment of second replication, PCR amplification were coducted prior to size selection and a range of fragment size (200-500 bp) was selected. Sequencing analysis was performed on the Illumina Genome Analyzer II according to the manufacturer's instructions.

Sequencing Platform

instrument_model
Illumina Genome Analyzer IIx

mm10

Number of total reads
30414897
Reads aligned (%)
95.7
Duplicates removed (%)
32.6
Number of peaks
23503 (qval < 1E-05)

mm9

Number of total reads
30414897
Reads aligned (%)
95.1
Duplicates removed (%)
32.5
Number of peaks
23523 (qval < 1E-05)

Base call quality data from DBCLS SRA