|
| Status |
Public on Mar 29, 2013 |
| Title |
infected blood endothelial cell (BEC) replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
BEC stably infected with rKSHV.219
|
| Organisms |
Homo sapiens; Human herpesvirus 8 strain rKSHV.219 |
| Characteristics |
infection: Human herpesvirus 8 strain rKSHV.219 (infection: stable)
|
| Growth protocol |
Microvascular LEC and BEC were cultured in EGM2-MV media (Lonza). Macrovascular HUVEC and HAEC were cultured in EGM2 media (Lonza). Cells stably infected with rKSHV.219 were maintained in 0.25 ug/mL puromycin (Invivogen).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
All samples were harvested in RLT buffer and RNA was isolated using the RNeasy Mini Kit according to the manufacturer’s protocol (Qiagen).
|
| Label |
Cy5
|
| Label protocol |
The Quick Amp Labeling Kit (Agilent) was used according to the manufacturer’s protocol to generate Cy5/Cy3 labeled cRNA from 450 ng of total RNA, which contains mix of spike-in transcripts A + B (Agilent). An equal amount of the spike-in transcript mix were added to both Cy5 and Cy3 labeled samples. The spike-in transcripts were detected by E1A probes on the array and used for linear normalization subsequent to the LOWESS normalization performed by the Agilent feature extraction software.
|
| |
|
| Channel 2 |
| Source name |
Common reference: mix of several different cell types (BJAB, BCBL, iSLK, iSLK.219), both latent and lytic.
|
| Organisms |
Homo sapiens; Human gammaherpesvirus 8 |
| Characteristics |
common reference composition: cell type: mix of several different cell types (BJAB, BCBL, iSLK, iSLK.219); virus: HHV8 Type M (in BCBL) or HHV8 Type P (in iSLK.219); infection status: uninfected (BJAB and iSLK) or stably infected (latent & lytic BCBL and iSLK.219)
|
| Growth protocol |
Microvascular LEC and BEC were cultured in EGM2-MV media (Lonza). Macrovascular HUVEC and HAEC were cultured in EGM2 media (Lonza). Cells stably infected with rKSHV.219 were maintained in 0.25 ug/mL puromycin (Invivogen).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
All samples were harvested in RLT buffer and RNA was isolated using the RNeasy Mini Kit according to the manufacturer’s protocol (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
The Quick Amp Labeling Kit (Agilent) was used according to the manufacturer’s protocol to generate Cy5/Cy3 labeled cRNA from 450 ng of total RNA, which contains mix of spike-in transcripts A + B (Agilent). An equal amount of the spike-in transcript mix were added to both Cy5 and Cy3 labeled samples. The spike-in transcripts were detected by E1A probes on the array and used for linear normalization subsequent to the LOWESS normalization performed by the Agilent feature extraction software.
|
| |
|
| |
| Hybridization protocol |
Samples were hybridized to the custom tiling microarrays according the manufacturer’s protocol (Agilent). Hybridized microarrays were washed according to the manufacturer’s protocol (Agilent).
|
| Scan protocol |
Microarrays were scanned on the GenePix 4000B scanner (Axon instruments) and all feature intensities collected using the GenePix Pro 6.0 software.
|
| Description |
inf BEC replicate 2
Biological replicate 2 of 2. All samples in Experiment 2 were processed in parallel, visualized on the same microarray on the same day, and the resulting microarray data was analyzed as one collective data set.
|
| Data processing |
TIFF images of scanned slides were analyzed using Feature Extraction Software version 9.5.3 (Agilent) using a custom grid file (017577_D_F_20070822). LOWESS normalized, background subtracted data were obtained from log2 of processed Red signal/processed Green signal. Agilent software was used. Data of identical probes were averaged. Data were subjected to a further linear normalization step by the average of the features detecting the spike-in transcripts. Only features that did not have any feature non-uniformity or population outlier flags contributed to the averages used for determining the normalization factor. Removed any data that failed any of the following spot quality filters: 1. gIsFound = 1 AND rIsFound = 1; 2. no PopnOL or NonUnifOL detected; 3. g OR r IsWellAboveBackground. Data of every probe was centered to the average of the corresponding control samples. Data for host genes were removed and probes against viral sequences were ordered by position in genome.
|
| |
|
| Submission date |
Mar 28, 2013 |
| Last update date |
Mar 29, 2013 |
| Contact name |
Henry H Chang |
| E-mail(s) |
changh88@gmail.com
|
| Organization name |
Novartis Institutes for BioMedical Research
|
| Department |
Infectious Diseases Group
|
| Street address |
4560 Horton Street
|
| City |
Emeryville |
| State/province |
CA |
| ZIP/Postal code |
94608 |
| Country |
USA |
| |
|
| Platform ID |
GPL16894 |
| Series (2) |
| GSE45591 |
Mock and stably infected rKSHV.219 cells (LEC, BEC, HUVEC, and HAEC) [Expt2] |
| GSE45600 |
A unique herpesviral transcriptional program in KSHV-infected lymphatic endothelial cells leads to mTORC1 activation and rapamycin sensitivity |
|
