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| Status |
Public on Feb 27, 2014 |
| Title |
RNAPII_MCF7 |
| Sample type |
SRA |
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| Source name |
MCF-7_RNAPII_ChIP-seq
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF-7
cell type: breast adenocarcinoma cells
chip antibody: RNAPII
chip antibody vendor: Covance
chip antibody cat. #: MMS-126R
chip antibody lot #: E12CF00481
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| Treatment protocol |
MCF7 cells were maintained in DMEM without phenol red and supplemented with 5% charcoal–dextran-treated FBS for 4 days prior to their use in estradiol (E2) treatment (10-7M, 30 min).
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| Growth protocol |
MCF7 cells were grown to 70% of confluence in DMEM with 10% FBS.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For ChIP and FAIRE assays, DNA was extracted according to Svotelis et al. (Methods Mol Biol., 2009) and to Eeckhoute et al. (Genome Res., 2009) with few modifications. In brief, ~20-30.106 cells were cross-linked using 1.1% formaldehyde for 10 or 30 min at RT in 20ml of PBS 1X, reaction was quenched with 1ml of 2.5M Glycine. Cells were lysed with SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris–HCI of pH 8.1) during 30 min at 4°C and sonicated to have DNA fragment size between 200 and 300 bp. For ChIP, lysate was incubated overnight at 4°C on a rotator with 5µg of antibody. 35 µl of protein-A magnetic beads (Invitrogen) were added during 2h before washing with TSE-150 (0.1% SDS, 1% Triton, 2 mM EDTA, 20 mM Tris–HCl of pH 8.1, 150 mM NaCl), TSE-500 (0.1% SDS, 1% Triton, 2 mM EDTA, 20 mM Tris of pH 8.1, 500 mM NaCl), LiCl detergent (0.25 M LiCl, 1% NP40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris of pH 8.1) and three times with TE 1X. For FAIRE, soluble chromatin was subjected to three consecutive phenol-chloroform extractions. DNA was then reverse-crosslinked overnight at 65ºC and treated with RNAse A for 30 min at 37°C, followed by Proteinase K for 2 h at 37°C. After purification, DNA was quantified with Picogreen (Invitrogen) before library preparation.
Libraries were prepared according to Illumina's instructions. Briefly, 10 ng (ChIP-seq) or 50 ng (FAIRE-seq) of DNA were end-repaired using a combination of T4 DNA polymerase and T4 polynucleotide kinase (Enzymatics). DNA fractions of ~300 bp were selectively isolated with solid-phase reversible immobilization (SPRI) beads (Agencourt AMPure, Beckman Coulter). After adapter ligation, DNA was PCR amplified with Illumina primers, the quality and quantity of each DNA library was analyzed using Agilent Bioanalyser before sequencing on HiSeq (Illumina) following the manufacturer's protocols.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Sample 4
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| Data processing |
Reads were aligned to the Human Reference Genome (assembly hg18) using BWA version 0.6.1 (Li and Durbin, 2010).
Only sequence reads that were uniquely mapped to the genome and with a MAQ mapping quality score >10 were used for the analysis.
Aligned tags were converted to WIG files by F-Seq (Boyle et al. 2008)
Genome_build: hg18
Supplementary_files_format_and_content: wig files containing aligned tags
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| Submission date |
May 16, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Nicolas Gévry |
| E-mail(s) |
nicolas.gevry@usherbrooke.ca
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| Organization name |
Université de Sherbrooke
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| Street address |
2500 boulevard de l'université
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| City |
Sherbrooke |
| State/province |
Québec |
| ZIP/Postal code |
J1K 2R1 |
| Country |
Canada |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE47027 |
LRH-1 governs vital transcriptional programs in endocrine sensitive and resistant breast cancer cells: LRH-1 ChIP-seq |
| GSE54892 |
LRH-1 governs vital transcriptional programs in endocrine sensitive and resistant breast cancer cells |
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| Relations |
| BioSample |
SAMN02146465 |
| SRA |
SRX278612 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1143125_RNAPII_MCF7.wig.gz |
176.5 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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