|
| Status |
Public on Jul 07, 2010 |
| Title |
SN_24hr_APAP_P32 |
| Sample type |
RNA |
| |
|
| Source name |
SN_24hr_APAP_P32
|
| Organism |
Mus musculus |
| Characteristics |
cell line: TAMH
media: TAMH cells were grown in serum-free Dulbecco’s modified Eagle’s medium/Ham’s F12 (1:1) media supplemented with 100nm dexamethasone, 10nM nicotinamide, 0.1% (v/v) gentamicin, and an ITS premix containing insulin (5mg/mL), transferrin (5mg/mL) and selenium (5ng/mL).
passage: Cell passages between 25 and 35 were grown in a humidified incubator with 5% CO2 and 95% air at 37ºC. During passages, cells were incubated for one minute with trypsin. Cell detachment was monitored using a microscope.
plating: Once detachment was complete, 5mL of 0.5mg/mL soybean trypsin inhibitor in Hank’s balanced salt solution was added before plating.
|
| Biomaterial provider |
The University of Washington (Seattle, WA).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from TAMH cells dosed with 2mM APAP, 2mM AMAP or control culture media for 2, 6 and 24-hour timepoints. For each treatment, cells were grown to confluence in two 150cm2 tissue culture dishes and dosed. At the end of each treatment, cells were harvested using a rubber scraper. Cell pellets were collected by centrifugation, and washed using ice-cold DPBS. Trizol reagent was added to the DPBS-washed pellet, vortexed and passed through a 22G needle multiple times to ensure complete cell lysis. Chloroform was then added, and the mixture was spun in a microcentrifuge at 8200g. The aqueous phase was isolated and dissolved in 70% ethanol. The resulting mixture was loaded onto a Qiagen RNeasy column (Valencia, CA). Purified total RNAs were eluted in sterile water according to the manufacturer’s protocol.
|
| Label |
biotin
|
| Label protocol |
For microarray hybridization, RNA was processed for hybridization to Affymetrix GeneChip® Mouse Gene 1.0 ST Arrays through a core facility in the Functional Genomics Laboratory at the Center for Ecogenetics and Environmental Health at the University of Washington according to the manufacturer’s instructions.
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| |
|
| Hybridization protocol |
The standard hybridization protocol was used as recommended by Affymterix.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneArray Scanner 3000 through the University of Washington Functional Genomics Laboratory at the Center for Ecogenetics and Environmental Health (http://depts.washington.edu/ceeh/ServiceCores/FC1/FC1.html).
|
| Description |
TAMH cells
|
| Data processing |
Affymetrix Expression Console v1.1 RMA normalization log2
|
| |
|
| Submission date |
Oct 18, 2009 |
| Last update date |
Sep 05, 2017 |
| Contact name |
James William MacDonald |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Washington
|
| Department |
Environmental and Occupational Health Sciences
|
| Street address |
4225 Roosevelt Way NE
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98105-6099 |
| Country |
USA |
| |
|
| Platform ID |
GPL6246 |
| Series (1) |
| GSE18614 |
Differential Regulation of Mitogen-Activated Protein Kinases by Acetaminophen in TAMH cells |
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