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Sample GSM798440 Query DataSets for GSM798440
Status Public on Jan 04, 2012
Title SLX-2574.433.s_2
Sample type SRA
 
Source name Human breast cancer cell line
Organism Homo sapiens
Characteristics condition: Tam-Responsive
tissue: MCF7
Growth protocol Cell culture: MCF-7, ZR75-1, T-47D and BT-474 human cell lines were obtained from ATCC. MCF-7 cells were grown in DMEM containing 10% heat-inactivated FBS, 2 mM L glutamine, 50 U/ml penicillin and 50 ug/ml streptomycin. ZR75-1, T-47D and BT-474 cells were grown in RPMI containing 10% heat-inactivated FBS, 2 mM L glutamine, 50 U/ml penicillin and 50 ug/ml streptomycin. To validate the cell lines, samples were genotyped by Health Protection Agency (http://www.hpa.org.uk) (MCF-7, ZR75-1, T-47D) or by in-house genotyping (BT-474). TAM-R cells17 were obtained from Dr Iain Hutcheson and Prof. Robert Nicholson (Cardiff) and were maintained in phenol red-free DMEM containing 5% charcoal dextran-treated FBS, 2 mM L glutamine, 50 U/ml penicillin and 50 ug/ml streptomycin and 10 nM 4-hydroxytamoxifen.
Primary tumour material: The ER+ breast cancer tumours were obtained from the Nottingham Tenovus primary breast cancer series, Addenbrooke's Hospital and Imperial Hospital Hammersmith, London, UK with appropriate ethical approval from the repositories. The malignant pericardial effusion and the two distant metastases were obtained from Imperial Hospital Hammersmith, London, UK.
Extracted molecule genomic DNA
Extraction protocol For ChIP in the tumours and metastases, the frozen sample was cut into smaller pieces and thawed in 1% (final concentration) formaldehyde for 20 minutes at room temperature. The reaction was quenched by adding 0.1 volume of 2M glycine for 10 minutes. The sample was disaggregated by Dounce homogenisation and processed according to standard ChIP procedures (Schmidt D et al, Methods, doi:S1046-2023(09)00047-4 [pii]). The DNA was subsequently amplified as previously described (Schmidt D et al, Methods, doi:S1046-2023(09)00047-4 [pii]). For the malignantpericardial effusion, epithelial cells were first enriched using Dynabeads conjugated with Epcam.
For ChIPs from cell line material, proliferating cells were cross-linked and processed for ChIP as previously described (Schmidt D et al, Methods, doi:S1046-2023(09)00047-4 [pii]). For the TAM-R cells, ER ChIP-seq was performed on cells grown in DMEM containing 10% FBS and 10 nM tamoxifen for 24 hours. For the cocktail experiments, cells weretreated with 100 ng/ml IGF-1, 100 ng/ml EGF, 1 ng/ml TNF-alpha and 10 ng/ml IL-6 for 90 minutes.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina Genome Analyzer IIx
 
Description Input DNA
MCF-7_Input
Data processing Alignments: Sequences generated by the Illumina Genome Analyzer were processed by the Illumina analysis pipeline version 1.6.1, and aligned to the Human Reference Genome (assembly hg18, NCBI Build36.1, March 2008) using BWA version 0.5.523. Reads were filtered by removing those with a BWA alignment quality score less than 15.
Peaks: Enriched regions of the genome were identified by comparing the ChIP samples to input samples using the MACS peak caller version 1.3.7.113. Additional peak calls were determined for the tumour samples using the SWEMBL peak caller version 3.2 (Wilder et. al, in prep) with default parameters except with -R = 0.005. For tumours without a corresponding input, all the available tumour input reads were combined and down sampled to derive acontrol track for peak calling purposes.
 
Submission date Sep 19, 2011
Last update date Oct 31, 2013
Contact name Suraj Menon
E-mail suraj.menon@cruk.cam.ac.uk
Organization name Cambridge Research Institute, Cancer Research UK
Street address Li Ka Shing Center, Robinson Way
City Cambridge
ZIP/Postal code CB2 0RE
Country United Kingdom
 
Platform ID GPL10999
Series (1)
GSE32222 Differential oestrogen receptor binding is associated with clinical outcome in breast cancer
Relations
BioSample SAMN02198115
SRA SRX371484

Supplementary data files not provided
Processed data provided as supplementary file
Raw data not provided for this record

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