|
| Status |
Public on Dec 01, 2006 |
| Title |
p63_ChIP-chip_B3, (+)ActD, chip 06 of 14 |
| Sample type |
genomic |
| |
|
| Source name |
Cervical carcinoma
|
| Organism |
Homo sapiens |
| Characteristics |
Age: 66 yrs Gender: Female Ethnicity: Caucasian
|
| Biomaterial provider |
Peter Howley, Harvard Medical School, Boston, MA
|
| Treatment protocol |
Cells were treated with 5nM Actinomycin D or ethanol (vehicle control) for 24-26 hrs before harvesting by trypsinization
|
| Growth protocol |
Cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
1. Trypsinized cells were placed into fresh media and cross-linked by the addition of formaldehyde.
2. Cells were then spun down and the cell pellet was then resuspended in micrococcal nuclease (MNase) reaction buffer.
3. The chromatin was then sonicated, spun down and the supernatant was diluted with IP-dilution buffer.
4. Diluted chromatin was precleared with protein A/G sepharose beads, and a portion reserved for control, or 'input', DNA (i.e. omitting immunoprecipitation).
5. The remaining chromatin was incubated with 4A4 anti-p63 antibody-coupled protein A/G sepharose beads.
6. Input and eluted material was de-crosslinked and purified.
|
| Label |
Biotin
|
| Label protocol |
Amplified ChIP (or matching input) DNA was fragmented with DNase I, and then biotin-labeled using terminal transferase (TdT).
|
| |
|
| Hybridization protocol |
Biotinylated DNA was hybridized to the Affymetrix tiled, human whole genome arrays, washed, and stained according to manufacturer's instructions.
|
| Scan protocol |
The microarrays were scanned using a custom-built Affymetrix scanner with a laser spot size of 3.5 um and a resolution (pixel size) of 1.0 um. Thus each feature on the array with the size of 5x5 um was represented by 25 data points (pixels). To estimate the intensity of each feature, the outer rim of pixels was discarded and the 75th-percentile of the remaining 9 pixels was used as the estimate of the intensity.
|
| Description |
Input and ChIP DNA was amplified by two rounds of primer extension (Round A) with random primers, followed by purification and two successive rounds of PCR (Round B and C).
The samples were then purified using column purification and ready for array hybridization protocols.
|
| Data processing |
A binding P-value for each genomic position was determined by the Wilcoxon rank sum test.
|
| |
|
| Submission date |
Oct 27, 2006 |
| Last update date |
Sep 02, 2008 |
| Contact name |
Kevin Struhl |
| Organization name |
Harvard Medical School
|
| Department |
Dept. Of Biological Chemistry & Molecular Pharmacology
|
| Lab |
Struhl lab
|
| Street address |
240 Longwood Ave.
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL4494 |
| Series (1) |
| GSE6132 |
Whole genome mapping of p63 binding sites in ME180 human cervical carcinoma cells |
|
