|
| Status |
Public on Jul 19, 2007 |
| Title |
HepG2-SRF_Human Tiling 2.0R A Array |
| Sample type |
genomic |
| |
|
| Source name |
HepG2 - hepatocarcinoma cell line
|
| Organism |
Homo sapiens |
| Characteristics |
chromatin immunoprecipitation on Affymetrix whole genome tiling arrays 2.0R (chip-chip) for transcription factor SRF in HepG2 cells (ChIP)
|
| Treatment protocol |
The background model was created using reverse crosslinked chromatin, instead of a ChIP, which was amplified and hybed to arrays with the same protocol as the ChIP samples. For their respective ChIPs, 5ug of antibody was used. Each of the three antibodies from Santa Cruz biotechnology: GABPa G1 (sc-28312), and rabbit polyclonal SRF G20 (sc-335). Antibody specificity had been previously validated by Western blot to demonstrate that the correct size protein was identified.
|
| Growth protocol |
We used a single growth of formaldehyde crosslinked HepG2 cells, which were pelleted and sent to Stanford on dry ice by the ENCODE common cell culture source (National Cell Culture Center) to produce chromatin according to standard procedures (after Johnson et al., 2007). 2x10^7 cells were used for each chromatin IP.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin extracted according to standard procedures (after Johnson et al., 2007).
|
| Label |
biotin
|
| Label protocol |
Each individual ChIP was amplified using a random primer amplification protocol (Cooper et al., 2007), except 2mM dUTP was added to the 10mM dNTP mix. Four ChIPs were pooled for each replicate array set. Two positive QPCR primers were used to QC each pool of IPs. Labeling thereafter was according to standard Affymetrix protocols.
|
| |
|
| Hybridization protocol |
Hybridization was performed according to manufacturer's instructions.
|
| Scan protocol |
According to standard Affymetrix protocols.
|
| Description |
chromatin immunoprecipitation on Affymetrix whole genome tiling arrays 2.0R (chip-chip) for transcription factor SRF in HepG2 cells (ChIP)
|
| Data processing |
We used MAT (Johnson et al., 2006) to process the raw CEL files. The following settings were used: BandWidth = 300, MaxGap = 300, MinProbe = 10, Trim = 0.1, pvalue=1e-5.
|
| |
|
| Submission date |
Jul 16, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
David Scott Johnson |
| E-mail(s) |
seasquirtdoctor@gmail.com
|
| Phone |
650-725-3018
|
| Organization name |
Stanford University
|
| Department |
Genetics
|
| Lab |
Richard M. Myers
|
| Street address |
300 Pasteur Drive
|
| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
| |
|
| Platform ID |
GPL4910 |
| Series (1) |
| GSE8489 |
ChIP-chip analysis of transcription factors GABP, SRF, TAF, and NRSF on Affymetrix whole genome tiling arrays 2.0R |
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