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| Status |
Public on Jan 05, 2011 |
| Title |
ERα sequencing in E2 (estradiol) treated MCF-7 cells |
| Sample type |
SRA |
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| Source name |
MCF-7 breast cancer cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF-7 breast cancer cells
fraction: ChIP DNA
antibody manufacturer: Santa Cruz
antibody catalog number: sc-543
antibody: ERα
agent: E2
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| Treatment protocol |
For ChIP assay, the details are as described previously (Pan et al 2008). The reagents were purchased from Sigma Aldrich unless otherwise specified. Briefly, cells were cultured for 3 days in phenol red-free DMEM/F12 (or RPM1640 for T47D) (Invitrogen) containing 5% charcoal-stripped FBS before 100nM E2 treatment (or mock treatment with ethanol) for 45 mins.
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| Growth protocol |
Human mammary cancer cell line MCF-7 and uterine cancer cell line Ishikawa were obtained from European Collection of Cell Culture (ECACC, Porton Down, Salisbury, SP4 0JG, UK). Human mammary cancer cell line T47D was obtained from the American Type Culture Collection (ATCC, Manassas, VA), uterine cancer cell line ECC1. MCF-7, ECC1 and Ishikawa cells were cultured in Dulbecco’s modified Eagle’s medium(DMEM, Invitrogen) supplemented with 5% fetal bovine serum(FBS, Invitrogen), penicillin-streptomycin(Invitrogen), and gentamycin (Invitrogen) in a 37 °C incubator at 5% CO2. T47D cells were maintained in RPM 1640 medium (Invitrogen) with same additives as the other cell lines.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The cells were then cross-linked with 1% (v/v) formaldehyde for 10 mins at room temperature and stopped with 125 mM glycine for 5 min. The crude nuclei were isolated with Triton X-100 lysis buffer ( 0.25% Triton X-100, 10 mM EDTA, 10 mM Tris-HCl (pH 8.1), 100 mM NaCl, 1x Protease Inhibitor (Roche) added freshly). The cross-linked cells were washed with phosphate-buffered saline, resuspended in SDS lysis buffer (1% SDS (Bio-Rad), 5 mM EDTA, 50 mM Tris-HCl (pH 8.1), 1x protease inhibitor), and sonicated for 10 mins in a Biorupter (Diagenode) to generate DNA fragments with size about 500bp. The chromatin was then diluted with buffer (1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 150 mM NaCl, 1x protease inhibitor), precleared with 5mM BSA before immunoprecitating with ER antibody (Santa Cruz,sc-543) and rec-protein A-sephaose 4B (Invitrogen) overnight. The sephaose beads were washed with washing buffer and eluted with elution buffer (1% SDS and 0.1M NaHCO3), and then purified with QIA quick PCR purification kit (QIAGEN). For ChIP-Seq library construction, we used the same procedure as mentioned above to generate chip DNA and then processed the samples according to manufacturer’s instructions (ChIP-Seq DNA Sample Prep Kit, Illumina). Briefly, ChIP DNA was blunted and ligated to sequencing adaptors. After PCR amplification, the fragments with size between 150-300bp were purified and used for sequencing on the Illumina Genome analyzer.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
ERα ChIP sequencing, after E2 treatment
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| Data processing |
Sequence tags (26 bp reads) were obtained from Solexa sequencing of the ChIP and control samples (input DNA). Image analysis was performed by Firecrest and base calling was performed using Bustard. Quality assessment from Illumina’s Genome Analyzer indicated that the sequencing reads were of high quality. The reads were aligned to the March 2006 assembly of the human genome (hg18) using ELAND. Approximately 68~72% of the reads were mapped uniquely to the genome. Reads that mapped to multiple locations in the genome were discarded. Furthermore in case the same tag was sequenced repeatedly, only one copy of the tag was retained, as repetitive tags usually arise from biases during ChIP-DNA amplification and sequencing library preparation which add noise to the data. Based on the average size of the sequenced ChIP DNA fragments, the tags were extended 200 bp 3’ from the tag mapping coordinates to mimic the actual fragments. Thus a profile of the ChIP fragments over the whole genome assembly was obtained. File format: chromosome
Peaks: High confidence ERBS were identified with peak finding algorithm described in supplementary methods with FDR of 0.1% (De Santa et al., 2009). These high confidence peaks were used for all subsequent analyses in this report. (De Santa et al, 2009) (Reference:De Santa F., Narang V., Yap Z.H., et al. (2009) Jmjd3 contributes to the control of gene expression in LPS-activated macrophages. EMBO J. 28: 3341-52). The identified peaks were filtered. First, an estimated peak intensity threshold based on a random distribution of tags over the genome was used to remove random low-intensity peaks. The FDR for a library was determined by a Monte Carlo simulation, in which extended to 3’ direction 200 bp fragments randomly extracted from the genome were used to estimate the numbers of random peaks with different intensity values. The number of random fragments was equal to the sequencing depth of each library under analysis. The minimum intensity that satisfies the 0.001 criterion is selected as the lower cut-off for calling confident peaks.
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| Submission date |
Aug 31, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Cheng Wei Chang |
| E-mail(s) |
[email protected]
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| Organization name |
GIS
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| Department |
Computational and Mathematical Biology 1
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| Street address |
Genome Institute of Singapore Genome #02-01 60 Biopolis Street
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| City |
Singapore |
| ZIP/Postal code |
138672 |
| Country |
Singapore |
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| Platform ID |
GPL9115 |
| Series (1) |
| GSE23893 |
Tissue-type specific estrogen signaling in breast and uterine cancer cells |
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| Relations |
| SRA |
SRX026530 |
| BioSample |
SAMN00110683 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM589237_SCS737_029_unique_hits.tags.bed.gz |
47.1 Mb |
(ftp)(http) |
BED |
| GSM589237_mcf7e2_peaks.txt.gz |
102.6 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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