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| Status |
Public on Jan 07, 2013 |
| Title |
DS018_MCF-7_foxm1_Thiostrepton_7 |
| Sample type |
SRA |
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| Source name |
MCF-7 breast adenocarcinoma cells, thiostrepton, FOXM1 ChIP
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| Organism |
Homo sapiens |
| Characteristics |
cell_line: MCF-7
cell_type: ER-positive breast adenocarcinoma cells
treatment: thiostrepton
chip_target: FOXM1
replicate: 7
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| Treatment protocol |
Cells were grown to 60% confluence, and then treated with Thiostrepton (10μM) or DMSO (0.1%) for 4 hr.
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| Growth protocol |
Human MCF-7 and MDA-MB-231 cell lines were obtained from the ECACC (European Collection of Animal Cell Cultures) and grown in DMEM supplemented with 10% FBS.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
6 x 15cm2 plates were used per IP protocol. IP was carried out as described in Schmidt et al. (2009) (PMID 19275939). In brief, cells were cross-linked using 1% formaldehyde for 10 min at RT, reaction was quenched with 1/20th volume 2.5M Glycine and cell pellets were collected after scraping plates. Nuclear lysates were prepared by lysing with LB1 (50mM Hepes-KOH, pH 7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton-X plus PI) 10 min at 4°C and LB2 (10mM Tris-HCl, pH8.0, 200mM NaCl, 1mM EDTA, 0.5M EGTA plus PI) 5 min at 4°C, followed by sonication in LB3 (10mM Tris-HCl, pH8.0, 100mM NaCl, 1mM EDTA, 0.5M EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine plus PI) using a diagnode sonicator to give a fragment size of ~200-300bp. Sonicated lysate was combined with 100ul protein-A magnetic beads preloaded with 10ug anti-FOXM1 antibody (Santa Cruz Biotechnology, USA; cat no. sc-502; lot no. D0110) O/N at 4°C. Beads were washed X6 in RIPA buffer (50mM HEPES pH7.6, 1mM EDTA, 0.7% Na deoxycholate, 1% NP-40, 0.5M LiCl) and DNA was eluted for 16 hr at 65°C in elution buffer (50mM Tris-HCl, pH8.0, 10mM EDTA, 1% SDS). Extracted DNA was treated with RNase A for 30 min at 37°C, followed by Proteinase K for 1 hr at 55°C, and DNA was purified by phenol-chloroform extraction. DNA was prepared for Illumina library construction by repair with Klenow and T4 polymerase, A-tailing with Klenow exo-polymerase and attachment of adapters (diluted 1:20) using DNA ligase. Adapter-ligated fragments were amplified by PCR using phusion polymerase (12 cycles), and ~200-300 bp fragments were extracted by gel purification. Library size and quantity were determined by Bioanalyzer.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Basecalls were performed using CASAVA version 1.7.
ChIP-Seq reads were aligned to the Human Reference Genome (assembly hg18, NCBI Build 36.6, March 2008) using bwa 0.6.1 with default settings.
Reads mapped with MAPQ < 15 were filtered out. Reads overlapping regions producing aspecific binding were also removed (regions obtained from http://hgdownload-test.cse.ucsc.edu/goldenPath/hg18/encodeDCC/wgEncodeMapability/wgEncodeDukeRegionsExcluded.bed).
Peaks were identified with MACS 1.4.1 using as a control file the input library matched by the same cell line as the treatment library.
Genome_build: hg18
Supplementary_files_format_and_content: Files produced by MACS that report peak positons and statistics. File description adapted from MACS manual (http://liulab.dfci.harvard.edu/MACS/00README.html): NAME_peaks.txt is a tab-delimited text file that contains information about called peaks. You can open it in Excel and sort/filter using Excel functions. Information includes: chromosome name, start position of peak, end position of peak, length of peak region, peak summit position related to the start position of peak region, number of tags in peak region, -10*log10(pvalue) for the peak region (e.g., pvalue =1e-10, then this value should be 100), fold enrichment for this region against random Poisson distribution with local lambda, FDR in percentage. Coordinates are 1-based, which is different from BED format.
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| Submission date |
Sep 10, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Dario Beraldi |
| E-mail(s) |
dario.beraldi@cruk.cam.ac.uk
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| Organization name |
Cambridge Research Institute
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| Street address |
Robinson Way
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| City |
Cambridge |
| ZIP/Postal code |
CB2 0RE |
| Country |
United Kingdom |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE40762 |
Genome-wide mapping of FOXM1 binding reveals co-binding with oestrogen receptor alpha in breast cancer cells (ChIP-seq) |
| GSE40767 |
Genome-wide mapping of FOXM1 binding reveals co-binding with estrogen receptor alpha in breast cancer cells |
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| Relations |
| SRA |
SRX185918 |
| BioSample |
SAMN01163933 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1001006_ds018_mcf7_ts.macs_peaks.txt.gz |
133.6 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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