|
| Status |
Public on Mar 05, 2015 |
| Title |
ChIP-seq_p65_E2+TNF_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
ChIP-seq_p65_E2+TNF
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF-7
cell type: ER Positive Breast Cancer Cells
treatment: E2+TNFa
chip antibody: p65 (Abcam ab7970)
|
| Treatment protocol |
Prior to all treatments, the cells were grown for 3 days in phenol red-free MEM Eagle medium supplemented with 5% charcoal-dextran-treated calf serum. All treatments were performed for 40 min with 100 nM 17β-estradiol, 25 ng/mL TNFα, or both.
|
| Growth protocol |
MCF-7 breast cancer cells were obtained from the ATCC and maintained in Minimum Essential Medium Eagle supplemented with 5% calf serum.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-seq libraries were generated from two biological replicates for each condition. The immunoprecipitated DNA was purified using a MiniElute PCR Purification Kit from Qiagen. After purification, 50 ng of ChIPed DNA for each condition was used to generate libraries for deep sequencing, as previously described (Quail et al., 2008), with some modifications. Briefly, the DNA was end-repaired and a single “A”-base overhang was added using the Klenow fragment of E. coli DNA polymerase. The A-modified DNA was ligated to Illumina sequencing adaptors using the Illumina TruSeq DNA Sample Prep Kit. The ligated DNA (250-300 bp) was size-selected by agarose gel electrophoresis and purified using a Qiagen Gel Extraction Kit.
The size-selected fragments were amplified using Illumina TruSeq P5 and P7 PCR primers, and purified using AMPure beads (Beckman Coulter). Quality control was performed to determine the size, purity, and concentration of the final libraries, which were sequenced using an Illumina HiSeq 2000 per the manufacturer’s instructions.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
ChIP-enriched DNA from MCF7 cells after 40min of E2+TNFα treatment
|
| Data processing |
Alignment: Short-reads were aligned to the human reference genome (hg19). Bowtie software package (Langmead et al., 2009) was used to align reads and the output was processed using custom Perl scripts, and imported into R for most the analysis.
Genome_build: hg19
Supplementary_files_format_and_content: bed
|
| |
|
| Submission date |
Oct 30, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
W. Lee Kraus |
| E-mail(s) |
lee.kraus@utsouthwestern.edu
|
| Organization name |
UT Southwestern Medical Center
|
| Street address |
5323 Harry Hines Blvd.
|
| City |
Dallas |
| State/province |
TX |
| ZIP/Postal code |
75390-8511 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE59530 |
TNFα Signaling Exposes Latent Estrogen Receptor Binding Sites in Breast Cancer Cells [ChIP-seq] |
|
| Relations |
| BioSample |
SAMN03153574 |
| SRA |
SRX747790 |
