|
| Status |
Public on Jan 11, 2012 |
| Title |
5_3 |
| Sample type |
RNA |
| |
|
| Source name |
nitrogen-limited cultures sparged with 20% oxygen, with 11 micromolar dissolved oxygen
|
| Organism |
Saccharomyces cerevisiae EC1118 |
| Characteristics |
condition: nitrogen-limited cultures sparged with 20% oxygen, with 11 micromolar dissolved oxygen
|
| Treatment protocol |
cultures were sparged with different oxygen/nitrogen mixtures to achieve different dissolved oxygen concentration. When reached the culture steady state for each level, samples were taken and inmediately placed on crushed ice. Afterwards, they were centrifuged at max. speed, eliminated the supernatant and the cell pellet frozen in liquid nitrogen and stored at -80ºC until analysis
|
| Growth protocol |
cells were grown in chemostats with a syntethic must medium (MS300), custom modified to achieve nitrogen limitation with d=0.1 h-1, pH=3.5
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from frozen cell pellets using the AxyPrep Multisource RNA kit (Axygen Biosciences, USA), modified for the use with glass beads for cell lysis. Briefly, we resuspended the cell pellet in the R-I kit buffer, added it to a screw-cap tube containing 250 µl of 500 µm acid-washed glass beads (Sigma, USA). We stirred the tubes in three cycles of 45 s in a Mini Bead BeaterTM (Biospec), standing an equivalent time in ice between cycles. Later, the lysate was centrifuged at 10.000 x g and supernatant was transferred to a tube with 250 µl of isopropanol. After this point, we followed the kit manufacturer instructions.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 500 ng total RNA (Expression Analysis Technical Manual, 2009, Affymetrix).
|
| |
|
| Hybridization protocol |
Following fragmentation, 15 ug of cRNA were hybridized for 16 hr at 45C on Yeast Genome 2,0 Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
|
| Scan protocol |
GeneChips were scanned using the Hewlett-Packard GeneChip Scanner 3000 7G.
|
| Data processing |
The data were analyzed in R with the affy, affyQCReport and simpleaffy packages from Bioconductor. Data was normalized using the RMA algorithm.
|
| |
|
| Submission date |
Jan 10, 2012 |
| Last update date |
Oct 01, 2012 |
| Contact name |
Felipe F Aceituno |
| E-mail(s) |
[email protected]
|
| Organization name |
PUC
|
| Department |
Chemical Engineering and Bioprocesses
|
| Lab |
Biotechnology
|
| Street address |
Vicuña Mackena 4860
|
| City |
Santiago |
| ZIP/Postal code |
45647687 |
| Country |
Chile |
| |
|
| Platform ID |
GPL2529 |
| Series (1) |
| GSE34964 |
Transcriptomic analysis of the oxygen response in Saccharomyces cerevisiae EC1118 under wine fermentation conditions |
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