|
| Status |
Public on Feb 15, 2013 |
| Title |
CEBPB_ATF4-GST_UM_HK_8-mer |
| Sample type |
protein |
| |
|
| Source name |
CEBPB and ATF4, HK design
|
| Organism |
Mus musculus |
| Characteristics |
dbd: BZIP
protein: CEBPB and ATF4
probe state: unmethylated
|
| Treatment protocol |
The double stranded arrays were either methylated (M) or unmethylated (UM). The methylation of the double stranded microarray were performed using 10 μl of M.SssI (New England Biolabs)in a total volume of 150μl at 37 °C for 3 hrs. The arrays were washed three times with PBS/0.5% Tween-20 and once with PBS.
|
| Extracted molecule |
protein |
| Extraction protocol |
We used 150 ng of plasmid DNA in a 16 μl in vitro transcription/ translation reaction using a PURExpress In Vitro Protein Synthesis Kit (New England BioLabs) supplemented with RNase inhibitor.
|
| Label |
Cy5
|
| Label protocol |
DBD sequences along with 15 amino acid residues on either side of the DBD in the native protein were cloned as NcoI-SacI fragments into a T7-driven C-terminal GST expression vector (Sharrocks 1994, Gene:138, 105-108).
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| |
|
| Hybridization protocol |
We used 150 ng of plasmid DNA in a 16 μl in vitro transcription/ translation reaction using a PURExpress In Vitro Protein Synthesis Kit (New England BioLabs) supplemented with RNase inhibitor. After a 2-h incubation at 37oC, 15 ml of the mix was added to 135 ml of protein-binding solution for a final mix of PBS/2% skim milk/0.2 mg per ml BSA/0.1% Tween-20. This mixture was added to an array previously blocked with PBS/2% skim milk and washed once with PBS/0.1% Tween-20 and once with PBS/0.01% Triton-X 100. After a 1-h incubation at room temperature, the array was washed once with PBS/0.5% Tween-20 and once with PBS/0.01% Triton-X 100. Cy5-labeled anti-GST antibody was added, diluted in PBS/2% skim milk. After a 1-h incubation at room temperature, the array was washed three times with PBS/0.05% Tween-20 and once with PBS.
|
| Scan protocol |
The array was imaged using an Agilent microarray scanner at 2 micron resolution. Images were scanned at two power settings: 100% photomultiplier tube (PMT) voltage (high), and 10% PMT (low). The two resulting grid images were then manually examined, and the scan with the fewest number of saturated spots was used. Image spot intensities were quantified using ImaGene software (BioDiscovery).
|
| Data processing |
We provide several scores for each 8-mer in each experiment. Z-Score – transformed kmer median intensity; E-score – Enrichment Score. E-scores are a modified version of AUC, and describe how well each kmer ranks the intensities of the spots. Please see the supplementary files on the platform record (GPL11260) to match the raw data files with the array probes.
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| |
|
| Submission date |
Feb 14, 2013 |
| Last update date |
Feb 15, 2013 |
| Contact name |
Ishminder Mann |
| Organization name |
National Cancer Institute, NIH
|
| Street address |
9000 Rockville pike
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL11260 |
| Series (1) |
| GSE44338 |
CG methylated microarrays identify novel methylated sequence bound by the CEBPB|ATF4 heterodimer that are active in vivo |
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