|
| Status |
Public on Aug 01, 2014 |
| Title |
pTH5480_HK_8mer_984 |
| Sample type |
protein |
| |
|
| Source name |
mybA_HK design
|
| Organism |
synthetic construct |
| Characteristics |
dbd: Myb_DNA-binding
platform_id_id design: HK
dbd source organism: Dictyostelium_discoideum
|
| Extracted molecule |
protein |
| Extraction protocol |
We used 150 ng of plasmid DNA in a 15 μl in vitro transcription/ translation reaction using a PURExpress In Vitro Protein Synthesis Kit (New England BioLabs) supplemented with RNase inhibitor and 50 μM zinc acetate.
|
| Label |
Cy5
|
| Label protocol |
DBD sequences along with 50 amino acid residues on either side of the DBD in the native protein were cloned as SacI–BamHI fragments into pTH5325, a modified T7-driven GST expression vector, in most cases. See paper supplemental web site (http://hugheslab.ccbr.utoronto.ca/supplementary-data/CisBP/) for exceptions.
|
| |
|
| Hybridization protocol |
We used 150 ng of plasmid DNA in a 15 μl in vitro transcription/ translation reaction using a PURExpress In Vitro Protein Synthesis Kit (New England BioLabs) supplemented with RNase inhibitor and 50 μM zinc acetate. After a 2-h incubation at 37oC, 12.5 ml of the mix was added to 137.5 ml of protein-binding solution for a final mix of PBS/2% skim milk/0.2 mg per ml BSA/50 μM zinc acetate/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-20/50 mM zinc acetate and once with PBS/0.01% Triton-X 100/50 mM zinc acetate. Cy5-labeled anti-GST antibody was added, diluted in PBS/2% skim milk/50 mM zinc acetate. After a 1-h incubation at room temperature, the array was washed three times with PBS/0.05% Tween-20/50 mM zinc acetate and once with PBS/50 mMzinc acetate.
|
| Scan protocol |
The array was imaged using an Agilent microarray scanner at 2 mM 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 |
Dec 17, 2013 |
| Last update date |
Aug 01, 2014 |
| Contact name |
Matthew Tyson Weirauch |
| E-mail(s) |
matthew.weirauch@cchmc.org
|
| Organization name |
Cincinnati Children's Hospital
|
| Street address |
3333 Burnet Avenue
|
| City |
Cincinnati |
| State/province |
OH |
| ZIP/Postal code |
45229 |
| Country |
USA |
| |
|
| Platform ID |
GPL11260 |
| Series (1) |
| GSE53348 |
Determination and Inference of Eukaryotic Transcription Factor Sequence Specificity |
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