|
| Status |
Public on Jun 12, 2013 |
| Title |
Yeast_Control_1h_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
exp growing S. cer treated with solvent (MeOH)
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: BY4743
growth phase: Exponential (OD600 1.0)
|
| Treatment protocol |
Yeast cultures at OD600 1.0 were treated with an arresting concentration of LoaOOH (75 µM) or an equivalent volume of methanol (absolute). Growth was then allowed to resume at 30 oC, with orbital shaking (150 rpm) for a further 60 min. A 50 mL aliquot for each replicate was centrifuged twice at 5000 g for 3 min, at room temperature. Cell pellets were snap-frozen in liquid nitrogen and stored at -80 °C until extraction.
|
| Growth protocol |
S. cerevisiae (BY4743) was grown in minimal medium, 30 oC, 150 rpm, from a single colony to an optical density of 1.0 (OD600 1.0). Six cultures were prepared in total, three biological replicates each for control and oxidant conditions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen cells were lysed with 1 mL cold (4 oC) TRIzol® reagent (Invitrogen) and mechanically disrupted with 0.75 g of acid washed glass beads (425-600 µm, Sigma) using a bead mill. Total RNA was then isolated according to the manufacturer’s instructions. Total RNA was further purified using a Qiagen Rneasy column purification kit according to the manufacturer's protocol.
|
| Label |
Biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol (GeneChip Expression Analysis Technical Manual, P/N 702232 Rev. 3, Affymetrix).
|
| |
|
| Hybridization protocol |
Following fragmentation, cRNA were hybridized for 16 h at 45 oC on GeneChip Yeast Genome 2.0 Arrays, Affymetrix. GeneChips were washed and stained with streptavidin-phycoerythrin conjugate in the Affymetrix FS450 fluidics station.
|
| Scan protocol |
Yeast_2 GeneChips were scanned using the Affymetrix GC3000 scanner.
|
| Description |
Yeast (OD600 1.0), grown from a single colony, were treated with either methanol or LoaOOH at an arresting concentration (75 µM) for 1 h. At 1h total RNA was isolated. Affymetrix Yeast_2 microarrays were used to probe changes in gene expression.
|
| Data processing |
Collected fluorescent intensity data (CEL files) were imported into the Partek® Genomic Suite™ version 6.5 (Partek inc.) with processing that included RMA normalisation and masking of all non-S. cerevisiae probes present on the Affymetrix Yeast_2 arrays.
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| |
|
| Submission date |
Jun 11, 2013 |
| Last update date |
Jun 12, 2013 |
| Contact name |
Patrick John O'Doherty |
| Organization name |
University of Western Sydney
|
| Street address |
Goldsmith Ave
|
| City |
Sydney |
| State/province |
NSW |
| ZIP/Postal code |
2560 |
| Country |
Australia |
| |
|
| Platform ID |
GPL2529 |
| Series (1) |
| GSE47820 |
Gene expression changes of Saccharomyces cerevisiae to linoleic acid hydroperoxide |
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