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Sample GSM798412

Query DataSets for GSM798412

Status

Public on Jan 04, 2012

Title

SLX-4120.667.s_3

Sample type

SRA

Source name

Human tumour tissue

Organism

Homo sapiens

Characteristics

condition: Responder
tissue: NT2289
antibody: none (input)

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
G8 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

May 15, 2019

Contact name

Suraj Menon

E-mail(s)

suraj.menon@cruk.cam.ac.uk

Organization name

Cambridge Research Institute, Cancer Research UK

Street address

Li Ka Shing Center, Robinson Way