|
| Status |
Public on Jun 04, 2019 |
| Title |
Zta_N182V_5mC_rep2 |
| Sample type |
protein |
| |
|
| Source name |
Zta(N182V), HK design
|
| Organism |
human gammaherpesvirus 4 |
| Characteristics |
dbd: bZIP
protein: Zta(N182V)
double-stranding treatment: 5-methylcytosine
|
| 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 |
The bZIP DNA binding domain (DBD) of wild type Zta was obtained from Dr. Timothy Hughes, University of Toronto, Canada as a GST construct cloned into a modified pDEST15 MAGIC vector (N-terminal GST) vector. Mutant constructs of Zta (N182S, N182Q, N182T, N182I, and N182V) were generated via site-directed mutagenesis of the wild-type construct (GenScript)
|
| Label |
Cy5
|
| Label protocol |
180 ng of plasmid containing DNA binding domains of Zta or Zta(N182) mutants were used to express proteins using PureExpress in-vitro transcription translation kit (NEB) in 25 µL reaction volume as per manufacturer’s instructions.
|
| |
|
| 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 three power settings: 100% photomultiplier tube (PMT) voltage (high), 50% PMT (medium), and 10% PMT (low). The 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(N182V) PBM experiment, 5mC
|
| 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 |
Jun 05, 2018 |
| Last update date |
Jun 05, 2019 |
| Contact name |
Desiree Tillo |
| E-mail(s) |
desiree.tillo@nih.gov
|
| Phone |
+1-240-760-7289
|
| Organization name |
NIH/NCI
|
| Street address |
41 Center Dr, Room D310
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL24408 |
| Series (1) |
| GSE115351 |
Mutating Zta (182N) changes sequence specific DNA binding (5mC + 5hmC) |
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