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Sample GSM1319627

Query DataSets for GSM1319627

Status

Public on Mar 21, 2016

Title

Input_control

Sample type

SRA

Source name

ER_LET-R_input

Organism

Homo sapiens

Characteristics

cell line: Let-R
cell type: Letrozole-resistant breast cancer cells

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 Genome Analyzer IIx

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