Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Unclassified
Antigen
Unclassified

Cell type

Cell type Class
Breast
Cell type
HMEC
Primary Tissue
Breast
Tissue Diagnosis
Normal

Attributes by original data submitter

Sample

source_name
Normal breast tissue
cell type
Primary human mammary epithelial cells (HMECs)
gender
female
genotype
BRCA1 mutation carrier

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Primary human breast epithelial cells were crosslinked with 1% formaldehyde at room temperature for 10 min, followed by incubation with 125 mM glycine for an additional 5 min. All following steps were carried out in buffers containing protease inhibitors in 4 °C until elution. Cells were pelleted by centrifugation at 1,000 g for 5 min, washed with PBS twice, then lysed in lysis buffer (5 mM HEPES, pH 7.9, 85 mM KCl, 0.5% Triton X-100) for 10 min. Nuclei were pelleted by centrifugation at 1,600 g for 5 min, and lysed in nuclei lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS). Chromosomal DNA was sonicated using a Bioruptor Pico to obtain < 300 bp fragments. 10% of sonicated DNA was saved as input, and the rest was incubated with H3K27ac antibody overnight (Abcam; ab4729). Dynabeads Protein A (Thermo Fisher Scientific; 10002D) was added the following day and incubated for additional 4 hr before washing. Washing was performed twice in TE sarcosyl buffer (50 mM Tris-HCl, pH 8.0, 2 mM EDTA, 0.2% sarcosyl), twice in TSE1 buffer (150 mM sodium chloride, 20 mM Tris-HCl pH 8.0, 2 mM EDTA, 0.1% SDS, 1% Triton-X-100), twice in TSE2 buffer (500 mM sodium chloride, 20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 0.1% SDS, 0.1% Triton X-100), twice in TSE3 buffer (250 mM lithium chloride, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1% sodium deoxycholate, 1% NP-40) and twice in TE buffer (50 mM Tris-HCl, pH 8.0, 2 mM EDTA). DNA was subsequently eluted from Dynabeads, reverse-crosslinked, and ethanol-precipitated. H3K27ac ChIP-seq libraries were constructed using a MicroPlex Library Preparation Kit (Diagenode; C05010011) following the manufacturer's guide. After a total of 10 cycles of PCR amplification, libraries were purified using Agencourt AMPure XP System (Beckman Coulter; A63880). Quality and quantity of the libraries were measured by a Qubit dsDNA HS Assay Kit (Life Technologies; Q32851) using a Bioanalyser 2100. Libraries with different index sequences were pooled together and then sequenced with a single-end 50 bp module using an Illumina Hiseq 3000 system. De-multiplexing was performed by CASAVA to generate FASTQ files for each sample. Between 38 and 92 million unique mapped reads were obtained for each sample.

Sequencing Platform

instrument_model
Illumina HiSeq 3000

hg38

Number of total reads
41910429
Reads aligned (%)
99.9
Duplicates removed (%)
2.3
Number of peaks
14794 (qval < 1E-05)

hg19

Number of total reads
41910429
Reads aligned (%)
99.9
Duplicates removed (%)
2.4
Number of peaks
14798 (qval < 1E-05)

Base call quality data from DBCLS SRA