|
| Status |
Public on Nov 11, 2024 |
| Title |
RORB.PBM14353.HK |
| Sample type |
protein |
| |
|
| Source name |
RORB.PBM14353.HK PBM, HK design
|
| Organism |
Homo sapiens |
| Characteristics |
design: HK
|
| Extracted molecule |
protein |
| Extraction protocol |
DBD sequences along with 50 amino acid residues on either side of the DBD in the native protein were cloned into pTH6838, a modified T7-driven GST expression vector. 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 to produce the proteins.
|
| Label |
Alexa 647
|
| Label protocol |
We used Alexa 647-labeled anti-GST antibody to detect the GST-tagged DNA binding proteins.
|
| |
|
| 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 37°C, 12.5 μL of the mix was added to 137.5 μL 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. Alexa 647-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 mM zinc acetate.
|
| Scan protocol |
The array was imaged using an Agilent microarray scanner at 2 uM 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. VALUE - median kmer intensity; 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.
Median intensity for the 8mer sequence
|
| |
|
| Submission date |
Aug 23, 2024 |
| Last update date |
Nov 11, 2024 |
| Contact name |
Timothy Hughes |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Toronto
|
| Department |
Terrence Donnelly Centre for Cellular and Biomolecular Research
|
| Lab |
Timothy Hughes Lab
|
| Street address |
160 College Street, Room 1350
|
| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5S 3E1 |
| Country |
Canada |
| |
|
| Platform ID |
GPL11260 |
| Series (1) |
| GSE275577 |
Perspectives on Codebook: sequence specificity of uncharacterized human transcription factors |
|
