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Sample GSM3176409 Query DataSets for GSM3176409
Status Public on Jun 04, 2019
Title Zta_N182Q_5mC_rep2
Sample type protein
 
Source name Zta(N182Q), HK design
Organism human gammaherpesvirus 4
Characteristics dbd: bZIP
protein: Zta(N182Q)
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(N182Q) 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.
 
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)

Data table header descriptions
ID_REF
VALUE 8-mer Z-score of the modified strand.

Data table
ID_REF VALUE
AAAAAAAA -0.813728798
AAAAAAAC -1.213360053
AAAAAAAG -0.661832756
AAAAAAAT -0.369794532
AAAAAACA -0.964720698
AAAAAACC -0.502703569
AAAAAACG -1.066888869
AAAAAACT -0.410480972
AAAAAAGA 2.139202596
AAAAAAGC -0.641941608
AAAAAAGG 1.448437261
AAAAAAGT 1.70702219
AAAAAATA 0.996365706
AAAAAATC 0.916801113
AAAAAATG -0.406864399
AAAAAATT -0.74139735
AAAAACAA -0.688957049
AAAAACAC -0.44212598
AAAAACAG -0.518978145
AAAAACAT -0.515361572

Total number of rows: 65536

Table truncated, full table size 1363 Kbytes.




Supplementary file Size Download File type/resource
GSM3176409_HK_535_D2_Zta_N182Q_5mC_rep2.raw_signal.txt.gz 1.1 Mb (ftp)(http) TXT
Processed data included within Sample table

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