|
| Status |
Public on Mar 13, 2008 |
| Title |
c-MYC localization in MCF7 (Whole Genome #6, Replicate #2) |
| Sample type |
genomic |
| |
|
| Source name |
Chromatin Immunoprecipitated DNA
|
| Organism |
Homo sapiens |
| Characteristics |
MCF7 cells. Anti-MYC antibodies were used for ChIP.
|
| Treatment protocol |
MCF7 Cells were E2-deprived for 3 days and then were treated with 10 nM E2 (45 minutes and 2 hours for mapping ER alpha and c-MYC binding sites, respectively) at 80% confluence. ~5x10^6 cells per ChIP were cross-linked with 1% formaldehyde for 10 minutes at 37oC then quenched with 125 mM glycine.
|
| Growth protocol |
MCF7 breast cancer cells (ATCC HTB-22) were grown in phenol-free Dulbecco's modified Eagles's medium, 10% fetal bovine serum, with supplemental bovine insulin (0.01 mg/ml), 2 mM L-glutamine, and antibiotics at 37oC in a 5% CO2 humidified atmosphere. Prior to hormone (E2 at 10 nM) or vehicle (100% EtOH) treatments, cells were changed to estrogen-depleted media for 72 hours by using 10% charcoal-stripped calf serum.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
According to the standard chromatin extraction protocol (Shang et al., 2000).
|
| Label |
biotin
|
| Label protocol |
ChIPed DNA was amplified using linker-mediated PCR amplification method. 2mM dUTP was added to the 10mM dNTP mix during the amplification.
|
| |
|
| Hybridization protocol |
According to standard Affymetrix protocols: Chromatin Immunoprecipitation Assay Protocol (http://www.affymetrix.com/support/technical/manuals.affx).
|
| Scan protocol |
According to standard Affymetrix protocols: Chromatin Immunoprecipitation Assay Protocol (http://www.affymetrix.com/support/technical/manuals.affx).
|
| Description |
Chromatin immunoprecipitation on Affymetrix whole genome tiling arrays 2.0R for transcription factor c-MYC in MCF7 cells (ChIP).
|
| Data processing |
Tiling array data were normalized and analyzed as described (Cawley et al, 2004). Briefly, raw data were normalized within ChIP and control groups and for each genomic position a local dataset composed of intensities for all adjacent probes within a window of ¡À250 bp was generated. A one-tailed Wilcoxon Rank Sum test was then applied to compare control and ChIP experiments and was performed in a sliding window across all tiled genomic regions. Significantly enriched probes (p < 1e-5) were locally extended by merging adjacent enriched probes within 100 bp and these merged regions were defined as transcription factor bound regions.
|
| |
|
| Submission date |
Mar 12, 2008 |
| Last update date |
Mar 12, 2008 |
| Contact name |
Sujun Hua |
| E-mail(s) |
shua@mdanderson.org
|
| Organization name |
MD Anderson Cancer Center
|
| Lab |
Ronald DePinho Lab
|
| Street address |
1901 East Rd
|
| City |
Houston |
| State/province |
Tx |
| ZIP/Postal code |
77054 |
| Country |
USA |
| |
|
| Platform ID |
GPL4915 |
| Series (1) |
| GSE10800 |
Genome-wide Binding Site Mapping of Estrogen Receptor alpha and c-MYC in Breast Cancer Cells |
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