|
| Status |
Public on Jan 01, 2020 |
| Title |
65k: HK_505_5hmC_A1_ZTA_N182T |
| Sample type |
protein |
| |
|
| Source name |
ZTA(N182T), HK design
|
| Organism |
Mus musculus |
| Characteristics |
dbd: bZIP
protein: ZTA(N182T)
double-stranding treatment: 5hmC
|
| Treatment protocol |
The single-stranded oligonucleotide microarrays were double-stranded by primer extension as described in Badis et al. Science (2009). Arrays were double-stranded using either cytosine, 5-methylcytosine (5mC, NEB) or 5-hydroxymethylcytosine (5hmC, Zymo Research). The resulting double-stranded DNA on the array thus contained probes with either cytosine on both strands or modified cytosine (5mC/5hmC) on one strand and cytosine on the second strand. This results in a hemi-methylated or hemi-hydroxymethylated state.
|
| Extracted molecule |
protein |
| Extraction protocol |
We used an N-terminal GST fusion construct of the wild-type construct of the DNA binding domain of Zta plus 50 flanking amino acids cloned into a modified pDEST15 MAGIC vector (Sharrocks, 1994). Mutant constructs of Zta(N182S, N182Q, N182I, N182V, N182T) were generated via site-directed mutagenesis of the wild-type plasmid (GenScript, USA).
|
| Label |
Cy5
|
| Label protocol |
Zta constructs were expressed using the PURExpress In vitro protein synthesis kit (NEB) according to the manufacturer’s protocol as previously described (Mann, 2013). Protein expression was monitored using a western blot anti GST HRP conjugated antibody and similar protein expression was confirmed by western blot.
|
| |
|
| Hybridization protocol |
The double-stranded arrays were blocked with 4% milk for 1 hour and washed with 0.1% Tween-20 in 1x PBS. Freshly synthesized protein (25 µL) was mixed with 125 µL of protein binding reaction mixture consisting of 4% milk in 1x PBS, 50 ng of salmon testes DNA and 0.2 µg/µL bovine serum albumin and added to double-stranded array. The protein binding reactions were carried out in hydration chamber for 1 hour followed by one wash with 0.5% Tween-20 in 1xPBS. The protein bound array was incubated with Alexa Fluor 647 conjugated Anti-GST antibody for 1 hour, followed by three washes with 0.05 % Tween-20. Finally array slides were washed and dried in 1x PBS and scanned using an Agilent Sure Scan II scanner.
|
| 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).
|
| Description |
ZTA(N182T) PBM experiment, 5hmC
|
| Data processing |
For all possible 65,536 8-mers, a transformed 8-mer median intensity (Z-score) was calculated from the median signal intensity across array probes containing each 8-mer. 8-mer Z-scores of the modified strand (the reverse complement of the array probe sequence) were used for downstream analyses.
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| |
|
| Submission date |
Feb 14, 2019 |
| Last update date |
Jan 02, 2020 |
| Contact name |
Charles Vinson |
| E-mail(s) |
vinsonc@dc37a.nci.nih.gov
|
| Organization name |
National Institutes of Health
|
| Department |
National Cancer Institute
|
| Lab |
Laboratory of Metabolism
|
| Street address |
37 Convent Drive
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20814 |
| Country |
USA |
| |
|
| Platform ID |
GPL11260 |
| Series (2) |
| GSE126588 |
Mutating Zta(N182) to S, Q, T, I, and V changes sequence specific DNA binding to four types of DNA (65k data set) |
| GSE126590 |
Mutating Zta(N182) to S, Q, T, I, and V changes sequence specific DNA binding to four types of DNA |
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