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| Status |
Public on Oct 21, 2009 |
| Title |
SMAD1/5, SMAD2 and SMAD4 binding/occupancy profiling in murine embryonic stem cells R1 |
| Organism |
Mus musculus |
| Experiment type |
Genome binding/occupancy profiling by array
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| Summary |
To determine the binding targets of SMAD1/5, SMAD2 and SMAD4 in mouse ESCs, ChIP combined with DNA microarray was carried out according to Agilent Mammalian ChIP-on-chip protocol. Briefly, immunoprecipitated (IP) DNA was blunted with T4 polymerase and ligated to linkers with T4 DNA ligase. For reference, 200 ng of input DNA was used. Ligated DNA was amplified with two rounds of PCR cycles. Two ug of each amplified sample were labeled with Cy5 for IP DNA, and Cy3 for input DNA using CGH labeling kit (Invitrogen). Four ug of each labeled sample was blocked with mouse Cot-1 DNA (Invitogen) and blocking agent (Agilent), and hybridized to DNA microarrays (G4490A, Agilent) for 40 h at 65℃. The microarrays were washed and stabilized with acetonitrile (Sigma), followed by stabilization and drying solution (Agilent). The arrays were scanned with Agilent DNA microarray scanner (G2565BA), and probe features were extracted using Feature Extraction Software (version 10.0). The raw data were processed by the R package limma and the log-ratio of IP over WCE was normalized within arrays using ‘loess’ method. And the normalized log-ratios were then used for SMAD target selection. Bound targets which was determined by the algorithm was validated by ChIP-stie specific PCR on a selected number of genes. The false positive rate was determined.
Keywords: ChIP-chip
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| Overall design |
ChIP assay were carried out in undifferentiated murine R1 ES cells and extracted DNA were analyzed by Agilent Mouse Promoter two-color Array(G4490A) according to manufacturer’s instruction. Anti-SMAD1/5 antibody generated with recombinant human SMAD1-linker (aa145-267)-His, Anti-SMAD2 antibody generated with recombinant human SMAD2-linker and Anti-SMAD4 antibody generated with recombinant human SMAD4-linker (aa144-316)-His were used for ChIP assay. Biological replicates: 1 SMAD1/5 replicate, 1 SMAD2 replicate and 1 SMAD4 replicate.
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| Contributor(s) |
Fei T, Xia K, Zhou B, Zhu S, Chen H, Li Z, Zhang J, Chen Z, Xiao H, Han JJ, Chen Y |
| Citation(s) |
19926752 |
| Submission date |
Oct 19, 2009 |
| Last update date |
Dec 06, 2012 |
| Contact name |
Bing Zhou |
| E-mail(s) |
bzhou@genetics.ac.cn
|
| Phone |
8610-64845797
|
| Fax |
8610-64845797
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| URL |
http://hanlab.genetics.ac.cn
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| Organization name |
Institute of Genetics and Developmental Biology, Chinese Academy of Sciences
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| Department |
Center for Molecular Systems Biology
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| Lab |
Laboratory of Jing-Dong Jackie Han
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| Street address |
Datun Road
|
| City |
Beijing |
| ZIP/Postal code |
100101 |
| Country |
China |
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| Platforms (2) |
| GPL4128 |
Agilent-014716 Mouse Promoter ChIP-on-Chip Set 244K, Microarray 1 of 2 (G4490A) (Feature Number version) |
| GPL4129 |
Agilent-014717 Mouse Promoter ChIP-on-Chip Set 244K, Microarray 2 of 2 (G4490A) (Feature Number version) |
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| Samples (6)
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| GSM462999 |
SMAD1/5 binding targets in murine R1 ES cells (1/2) |
| GSM463000 |
SMAD1/5 binding targets in murine R1 ES cells (2/2) |
| GSM463001 |
SMAD4 binding targets in murine R1 ES cells (1/2) |
| GSM463002 |
SMAD4 binding targets in murine R1 ES cells (2/2) |
| GSM593667 |
SMAD2 binding targets in murine R1 ES cells (1/2) |
| GSM593670 |
SMAD2 binding targets in murine R1 ES cells (2/2) |
|
| Relations |
| BioProject |
PRJNA121397 |
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