 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Mar 21, 2016 |
| Title |
ER_LET-R_Veh_01 |
| Sample type |
SRA |
| |
|
| Source name |
ER_LET-R_Veh
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Let-R
cell type: Letrozole-resistant breast cancer cells
treated with: 10% ethanol (vehicle control) for 45min
chip antibody: anti-ER
chip antibody vendor: Santa Cruz Biotechnologies
chip antibody cat. #: sc-543
|
| Treatment protocol |
Cells were maintained in steroid depleted medium for 72 hours prior to treatment with androstenedione (10-7 M; Sigma Aldrich, St. Louis, MO, USA) or Vehicle (10% ethanol) for 45minutes.
|
| Growth protocol |
Estrogen receptor positive MCF-7 breast cancer cells were obtained from American Type Culture Collection (ATCC). MCF-7 cells were stably transfected with the aromatase gene (CYP19) (Invitrogen, Paisley, UK) to generate aromatase inhibitor sensitive cells (Aro cells). Letrozole-resistant (LetR) cells were then generated by long-term (>3 months) culture of Aro cells with letrozole (10-6M; Novartis, Switzerland) and androstenedione (25 x 10-5 mol/L; Sigma Aldrich) in MEM supplemented with 10% charcoal-dextran-stripped FCS, 1% L-Glutamine, 1% Pen/Strep, and 200 mg/mL Geneticin (G418, Gibco Invitrogen). All cells were maintained at 37degreesCelcius, 5% CO2 in a humidified incubator.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were treated and harvested for ChIP-seq as described previously (Schmidt et al, 2009, http://www.ncbi.nlm.nih.gov/pubmed/19275939). The anti-ER (sc-543) from Santa Cruz Biotechnologies was used as the antibody. Chromatin was extracted from the cells and the protein of interest, along with the DNA attached, was immunoprecipitated using the antibody attached to Dynal beads (Dynabeads® Protein A 10001D, Life Technologies). The proteins were then removed from the DNA by reverse crosslinking overnight and the DNA purified and amplified before being sequenced.
DNA libraries were generated from ChIP output DNA by adaptor ligation, gel purification, and cycles of PCR as described by Illumina.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
The DNA sequencing was carried out on the Illumina GAIIx and Illumina HiSeq 2000
ChIP-seq reads were aligned to the hg19 genome using Bowtie (v0.12.9) with parameters "-m 1 -v 2 --strata --best"
Peak calling was carried out by MACS2 with a p-value cut off of 1e-03 and by linearly scaling the smaller dataset to the larger dataset (via the "--to-large" parameter).
Using the MACS2 results, a reliable peakset was produced by applying the Irreproducible Discovery Rate tool to the replicates of the androstenedione treated and vehicle samples. The peak sets for the androstenedione and vehicle samples were then assessed for overlapping peaks using bedtools (by at least 1bp).
This produced three peaks lists : 1) Androstenedione only peaks, 2) Vehicle only peaks and 3) Overlapping peaks between Androstenedione and Vehicle peaksets. They are represented as bed files
Genome_build: hg19
Supplementary_files_format_and_content: There are the ChIP-seq data files in bedgraph format from the MACS2 peak calling software.And there are bed files for the three peak lists: 1) Androstenedione only peaks, 2) Vehicle only peaks and 3) Overlapping peaks between Androstenedione and Vehicle peaksets
|
| |
|
| Submission date |
Jan 31, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Leonie Young |
| Organization name |
Royal College of Surgeons, Ireland
|
| Department |
Surgery
|
| Lab |
Endocrine Oncology Research Group
|
| Street address |
York Street
|
| City |
Dublin |
| ZIP/Postal code |
Dublin 2 |
| Country |
Ireland |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE54592 |
ER ChIP-seq of Androstenedione treated Letrozole Resistant Breast Cancer Cell line |
|
| Relations |
| BioSample |
SAMN02614520 |
| SRA |
SRX457214 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1319625_ER_LET-R_Veh_01_pileup.bedGraph.gz |
198.7 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
|
|
|
|
 |
