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| Status |
Public on Apr 19, 2019 |
| Title |
BSC122L: H3K27ac_NC_2 |
| Sample type |
SRA |
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| Source name |
Normal breast tissue
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Primary human mammary epithelial cells (HMECs)
gender: female
genotype: Non-carrier
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| Extracted molecule |
genomic DNA |
| Extraction 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.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 3000 |
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| Data processing |
H3K27ac ChIP-seq was aligned to the human genome by BWA and only unique mapped reads were saved. BELT, a bin based peak calling algorism that applies a statistical method to control false discovery rate (FDR), was used to call peaks. Super-enhancers were identified using ROSE.
Genome_build: hg19
Supplementary_files_format_and_content: bed files with super enhancers
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| Submission date |
Oct 15, 2018 |
| Last update date |
Apr 19, 2019 |
| Contact name |
Rong Li |
| Organization name |
University of Texas Health Science Center at San Antonio
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| Department |
Molecular Medicine
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| Street address |
7703 Floyd Curl Drive
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| City |
San Antonio |
| State/province |
TX |
| ZIP/Postal code |
78229 |
| Country |
USA |
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| Platform ID |
GPL21290 |
| Series (1) |
| GSE121229 |
BRCA1 Mutations Attenuate Super-Enhancer Function and Chromatin Looping in Haploinsufficient Human Breast Epithelial Cells |
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| Relations |
| BioSample |
SAMN10240648 |
| SRA |
SRX4883620 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3428172_BSC122L_SuperEnhancers.bed.gz |
1.6 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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