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Status |
Public on Jan 12, 2012 |
Title |
MEF_STAT5-KO-GH-IgG |
Sample type |
SRA |
|
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Source name |
MEF_STAT5-KO-GH-IgG
|
Organism |
Mus musculus |
Characteristics |
background strain: C57BL/6 genotype/variation: STAT5 KO; Stat5-/- tissue origin: 12.5-13.5-day fetuses cell type: mouse embryonic fibroblasts (MEFs) passages: 9-10 stimulated with: 1 ug/ml growth hormone (GH) for 45 min chip antibody: IgG control chip antibody vendor: R&D System chip antibody cat. #: AB-105C
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Treatment protocol |
After 5 hours starvation in serum free medium with 0.1% of BSA, MEFs were treated with growth hormone for indicated time period.
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Growth protocol |
Stat5+/- mice were bred into the C57BL/6 background. Stat5+/- mice were intercrossed and mouse embryonic fibroblasts (MEFs) were isolated from 12.5-13.5-day fetuses. MEFs were expanded in DMEM with high glucose supplements and 15% fetal bovine serum (FBS). MEFs of passage 9-10 were used for experiments.
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Extracted molecule |
genomic DNA |
Extraction protocol |
After 5-hour starvation, WT, Stat5-/-, or STAT5A-Stat5-/- MEFs were stimulated with or without 1 ug/ml GH for 45 min. MEFs were then cross-linked with 1% formaldehyde for 10 min. Chromatin from 5 × 10^6 cells was used for each ChIP experiment. Antibodies against STAT5 (sc-835, Santa Cruz, California) and IgG (AB-105C, R&D System) were used. The ChIP DNA fragments were blunt-ended, ligated to the Solexa paired-end adaptors, and sequenced with the Illumina Hi-seq 2000 genome analyzer.
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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 |
Experimental data sets came from wild type (WT-GH), Stat5-/- (KO-GH) and Stat5-/-overexpressing STAT5A (OE-GH) MEFs upon GH stimulation. The mapped tags of STAT5 antibodies and IgG samples were analyzed using the MACS program (http://liulab.dfci.harvard.edu/MACS/) with the following parameters; -mfold = 10,20 -g mm -p 1.0e-4 (p-value cutoff). The identified peaks were further split by using PeakSplitter (http://www.ebi.ac.uk/bertone/software.html). ChIP-seq data obtained from GH-treated Stat5-/- MEFs (Transfected with an empty vector, KO-empty-GH) served as negative controls and were independently processed with the same parameters. We utilized the GAS motif information to accurately identify bona fide STAT5 binding sites. The FIMO DNA motif identification tool (http://meme.nbcr.net/meme/cgi-bin/fimo.cgi) was used to scan the peak regions with a STAT5 position-specific scoring matrix (p-value threshold: 1e-3). The peaks including at least one GAS motif within their peak regions were regarded as bona fide STAT5 binding and used in this study.
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Submission date |
Jan 11, 2012 |
Last update date |
May 15, 2019 |
Contact name |
Keunsoo Kang |
E-mail(s) |
kangk1204@gmail.com
|
Phone |
82-41-550-3456
|
Organization name |
Dankook University
|
Department |
Microbiology
|
Lab |
Computational Biology Lab
|
Street address |
Dandae-ro 119
|
City |
Cheonan |
State/province |
Chungnam |
ZIP/Postal code |
31116 |
Country |
South Korea |
|
|
Platform ID |
GPL13112 |
Series (1) |
GSE34986 |
Genome-wide analyses reveal the extent of opportunistic STAT5 binding that does not yield transcriptional activation of neighboring genes. |
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Relations |
SRA |
SRX115365 |
BioSample |
SAMN00771533 |