|
| Status |
Public on Jul 12, 2017 |
| Title |
cluster-rep3 |
| Sample type |
RNA |
| |
|
| Source name |
TIVE cells,infected with Δcluster-KSHV,technical replicate 3
|
| Organism |
Homo sapiens |
| Characteristics |
tissue culture cell line: telomerase-immortalized vein endothelial cells
virus: Δcluster-KSHV, latent infection
|
| Treatment protocol |
Latently-infected TIVE cells were maintained in the presence of hygromycin (10 µg/mL) to prevent loss of KSHV episomes.
|
| Growth protocol |
TIVE cells were grown in complete Medium-199 (1% Pen-Strep, 20% FBS), supplemented with Endothelial cell growth supplement (Sigma E2759).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated with RNA-Bee (Tel-Test Inc.) using the protocol provided by the manufacturer. Total RNA (5-10 µg) was treated with DNase I (NEB) according to the manufacturer’s instructions and ethanol precipitated overnight.
|
| Label |
Cy3
|
| Label protocol |
RNA quantity and quality were measured by NanoDrop ND-1000 and RNA integrity was assessed by standard denaturing agarose gel electrophoresis. mRNA was purified from total RNA after removal of rRNA (mRNA-ONLY™ Eukaryotic mRNA Isolation Kit, Epicentre). Then, each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3’ bias utilizing a random priming method. The labeled cRNAs were purified by RNeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000.
|
| |
|
| Hybridization protocol |
1 μg of each labeled cRNA was fragmented by adding 5 μl 10 × Blocking Agent and 1 μl of 25 × Fragmentation Buffer, then heated the mixture at 60 °C for 30 min, finally 25 μl 2 × GE Hybridization buffer was added to dilute the labeled cRNA. 50 μl of hybridization solution was dispensed into the gasket slide and assembled to the LncRNA expression microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven. The hybridized arrays were washed, and fixed. and scanned with using the Agilent DNA Microarray Scanner (part number G2505C).
|
| Scan protocol |
The hybridized arrays were scanned with the Agilent DNA Microarray Scanner (part number G2505C).
|
| Description |
LncRNA expression of cells latently infected with Δcluster-KSHV.
|
| Data processing |
Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed using the GeneSpring GX v11.5.1 software package (Agilent Technologies). After quantile normalization of the raw data, LncRNAs with at least 3 out of 9 samples having flags in Present or Marginal (“All Targets Value”) were chosen for further data analysis. Differentially expressed LncRNAs with statistical significance between two groups were identified through Volcano Plot filtering. Hierarchical Clustering was performed using the Agilent GeneSpring GX software (version 11.5.1).
|
| |
|
| Submission date |
Oct 24, 2016 |
| Last update date |
Jul 12, 2017 |
| Contact name |
Rolf F Renne |
| E-mail(s) |
rrenne@ufl.edu
|
| Organization name |
University of Florida
|
| Department |
Molecular Genetics & Microbiology
|
| Lab |
CGRC 375
|
| Street address |
2033 Mowry Road
|
| City |
Gainesville |
| State/province |
FL |
| ZIP/Postal code |
32610 |
| Country |
USA |
| |
|
| Platform ID |
GPL16956 |
| Series (1) |
| GSE89114 |
Deregulation of lncRNAs by Kaposi's sarcoma-associated herpesvirus |
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