|
| Status |
Public on Oct 01, 2010 |
| Title |
BART-complemented cells, replicate 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
S1-1 cells
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: Experimental cells. They were transduced with retrovectors encoding the BART miRNAs, induced for dnEBNA1 expression with doxycycline, and cultured 20 days to evict the virus.
cell line: S1-1
condition: EBV evicted with dnEBNA1, BART miRNAs expressed ectopically
|
| Growth protocol |
Cells were diluted to 3-5x104 cells/mL in culture medium and treated with either 10 ng/mL doxycycline or the vehicle, ethanol. Live cell concentrations were measured every two or four days with a hemocytometer. Cells stained with trypan blue (with a 1:10 dilution of 0.3% trypan blue dissolved in 1x PBS) or exhibiting an aberrant morphology were considered non-viable. After counting, in necessary cells were diluted in fresh medium back to approximately the starting concentration (3-5x104 cells/mL) and doxycycline or ethanol was added.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using TRIzol reagent (Invitrogen), following the manufacturer’s protocols, except RNA precipitation was conducted in the presence of approximately 5 µg/mL linear acrylamide (Ambion) to enhance efficiency of precipitation as previously described (Gaillard and Strauss, 1990). The isolated RNA was then treated with Turbo DNAse (Ambion) following the manufacturer’s instructions and re-purified with TRIzol.
|
| Label |
cy3
|
| Label protocol |
Labelled cRNA was generated using Agilent's Quick Amp Labeling Kit following the manufacturer's instructions. This procedure uses T7 RNA polymerase, which both amplifies target material and incorporates cyanine 3- or cyanine 5-labeled CTP.
|
| |
|
| Channel 2 |
| Source name |
S1-1 cells
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: Control cells. They were transduced with empty retrovectors, mock induced (dnEBNA1 kept off), and cultured 20 days.
cell line: S1-1
|
| Growth protocol |
Cells were diluted to 3-5x104 cells/mL in culture medium and treated with either 10 ng/mL doxycycline or the vehicle, ethanol. Live cell concentrations were measured every two or four days with a hemocytometer. Cells stained with trypan blue (with a 1:10 dilution of 0.3% trypan blue dissolved in 1x PBS) or exhibiting an aberrant morphology were considered non-viable. After counting, in necessary cells were diluted in fresh medium back to approximately the starting concentration (3-5x104 cells/mL) and doxycycline or ethanol was added.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using TRIzol reagent (Invitrogen), following the manufacturer’s protocols, except RNA precipitation was conducted in the presence of approximately 5 µg/mL linear acrylamide (Ambion) to enhance efficiency of precipitation as previously described (Gaillard and Strauss, 1990). The isolated RNA was then treated with Turbo DNAse (Ambion) following the manufacturer’s instructions and re-purified with TRIzol.
|
| Label |
cy5
|
| Label protocol |
Labelled cRNA was generated using Agilent's Quick Amp Labeling Kit following the manufacturer's instructions. This procedure uses T7 RNA polymerase, which both amplifies target material and incorporates cyanine 3- or cyanine 5-labeled CTP.
|
| |
|
| |
| Hybridization protocol |
Scanned on an Agilent G2565BA scanner.
|
| Scan protocol |
Images were recorded and quantified using Agilent’s Scan Control software, version A. 7.0.1 (includes XDR functionality) and Agilent Feature Extraction software (v. 9.5.3).
|
| Description |
BART miRNA complemented loss of EBV, replicate 1 of 3
|
| Data processing |
Agilent Feature Extraction software (v. 9.5.3) was used with the manufacturer's default settings.
|
| |
|
| Submission date |
Jun 26, 2010 |
| Last update date |
Jun 29, 2010 |
| Contact name |
Bill Sugden |
| E-mail(s) |
sugden@oncology.wisc.edu
|
| Phone |
608-262-6697
|
| Organization name |
University of Wisconsin-Madison
|
| Department |
Oncology
|
| Street address |
1400 University Avenue
|
| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53706-1599 |
| Country |
USA |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE22586 |
The forced loss of Epstein Barr virus from Burkitt's lymphoma cells: uncomplemented cells vs BART miRNA complemented cells. |
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