|
| Status |
Public on Jan 01, 2013 |
| Title |
CREB1-array7 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
CREB1 ChIP
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: transgenic MCF7
transcription factor: CREB1
tag: modified LAP (localization and affinity purification) tag containing GFP
chip antibody: Goat anti-GFP
|
| Treatment protocol |
According to the standard chromatin extraction protocol (Shang et al., 2000).
|
| 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.
For each NR with known ligand, MCF7 Cells were hormone-deprived for 3 days and then were treated with 100 nM NR ligand 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. For orphan NRs and NR-cofactors, no ligand was used and ChIP was performed with cells growing in phenol-free Dulbecco's modified Eagles's medium, 10% fetal bovine serum, with supplemental bovine insulin (0.01 mg/ml).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIPed DNA was amplified using linker-mediated PCR amplification method. 2mM dUTP was added to the 10mM dNTP mix during the amplification.
|
| 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.
|
| |
|
| Channel 2 |
| Source name |
Input DNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: transgenic MCF7
|
| Treatment protocol |
According to the standard chromatin extraction protocol (Shang et al., 2000).
|
| 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.
For each NR with known ligand, MCF7 Cells were hormone-deprived for 3 days and then were treated with 100 nM NR ligand 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. For orphan NRs and NR-cofactors, no ligand was used and ChIP was performed with cells growing in phenol-free Dulbecco's modified Eagles's medium, 10% fetal bovine serum, with supplemental bovine insulin (0.01 mg/ml).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIPed DNA was amplified using linker-mediated PCR amplification method. 2mM dUTP was added to the 10mM dNTP mix during the amplification.
|
| 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 |
Ch2 input ctrl CEL files: input1-7.CEL, input2-7.CEL, input3-7.CEL
CREB1 ChIP - Array 7
|
| Data processing |
Tiling array data were normalized and analyzed as described (Hua et al, 2009). 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 +/-250 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-3) were locally extended by merging adjacent enriched probes within 100 bp and these merged regions were defined as transcription factor bound regions. Peaks located in the BAC regions, overlaps with GFP peaks, and overlaps with repetitive regions in the genome are filtered.
|
| |
|
| Submission date |
Nov 01, 2012 |
| Last update date |
Jan 01, 2013 |
| Contact name |
Jie Zhou |
| E-mail(s) |
jiezhou@uchicago.edu
|
| Phone |
3123163491
|
| Organization name |
University of Chicago IGSB
|
| Street address |
900 East 57th Street, KCBD 10100
|
| City |
Chicago |
| State/province |
ILLINOIS |
| ZIP/Postal code |
60637 |
| Country |
USA |
| |
|
| Platform ID |
GPL4916 |
| Series (1) |
| GSE41995 |
ChIP-chip of 38 nuclear receptors and NR co-factors in breast cancer cell line MCF7 |
|
