|
| Status |
Public on Jul 07, 2010 |
| Title |
HUVEC replicate 1 (P-type strain study) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
HUVEC replicate 1
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: HUVEC
virus: N/A
infection status: Mock
|
| 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 450ng of total RNA, which contains mix of spike-in transcripts A + B (Agilent). An equal amount of hte spike-in transcript mix are added to both Cy5 and Cy3 labeled samples. The spike-in transcripts are detected by E1A probes on the array and are used for a 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, SLK, HFF, HUVEC, TIME), both infected and uninfected.
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: mix of several different cell types (BJAB, BCBL, SLK, HFF, HUVEC, TIME)
virus: All infections were performed with either BCBL-1-derived KSHV or rKSHV.219.
infection status: both infected and uninfected
|
| 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 450ng of total RNA, which contains mix of spike-in transcripts A + B (Agilent). An equal amount of hte spike-in transcript mix are added to both Cy5 and Cy3 labeled samples. The spike-in transcripts are detected by E1A probes on the array and are used for a 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.
|
| 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 obtained from log2 of processed Red signal/processed Green signal. Agilent software was used.
Data of identical probes were averaged.
Data were subject 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.
Remove 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 |
May 24, 2010 |
| Last update date |
Jul 07, 2010 |
| Contact name |
Sanjay Chandriani |
| E-mail(s) |
[email protected]
|
| Organization name |
UCSF
|
| Street address |
513 Parnassus Avenue
|
| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94143 |
| Country |
USA |
| |
|
| Platform ID |
GPL10083 |
| Series (1) |
| GSE21972 |
Mock or KSHV cells (BJABJ, BCBL, TIME and HUVEC) vs common reference (mixture of RNA from both infected and uninfected cells) |
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