|
| Status |
Public on Jan 14, 2011 |
| Title |
PE-2 bottom of the fermentor x PE-2 fermentation, Replicate 2 (dye swap) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
PE-2 bottom of the fermentor, labeled with Cyanine-5
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: PE-2
fermentation stage: bottom of the fermentor (before feeding)
|
| Treatment protocol |
The sampling of wine containing yeast cells was made in three steps: after acid treatment (before feeding), termination of must feeding (three hours) and at the end of fermentation (nine hours). In each step, 1 mL of sample was collected from each fermentation flask. The samples were immediately frozen in liquid nitrogen and stored at ultra low temperature (-70oC) for further analysis of the transcriptome.
|
| Growth protocol |
The fed-batch fermentations with yeast cell recycle were conducted in bench scale reproducing all steps that occur in a typical industrial process of alcoholic fermentation in Brazil. Fermentations were carried out in triplicate for each strain. After centrifugation, the yeast cells were resuspended in water and fed with sugar cane must containing 18% sugars, being 50% from molasses and 50% from sugar cane juice. Fermentations were conducted at 33oC for 9 hours containing 10% of yeast cells (w / v). At the end of the first cycle, fermentative yeasts were centrifuged again and the cream of yeast treated with sulfuric acid to reach pH 2.5 for 1.5 hours at room temperature. After the acid treatment, yeast cells were fed with sugar cane must to start a new fermentation. In the second cycle, fermentation samples were collected for analysis of the transcriptome.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol following manufacturer's instructions, followed by DNAse treatment and purification using the RNeasy Mini Kit (Qiagen).
|
| Label |
Cy5
|
| Label protocol |
Prior to labelling, cDNA was synthesized from 5 µg of RNA using Agilent's cDNA Master Mix. cRNA amplification and labeling were performed by adding to the samples the Agilent Transcription Master Mix plus Cyanine-3 or Cyanine-5 for 2 hours at 40 oC.
|
| |
|
| Channel 2 |
| Source name |
PE-2 fermentation, labeled with Cyanine-3
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: PE-2
fermentation stage: fermentation
|
| Treatment protocol |
The sampling of wine containing yeast cells was made in three steps: after acid treatment (before feeding), termination of must feeding (three hours) and at the end of fermentation (nine hours). In each step, 1 mL of sample was collected from each fermentation flask. The samples were immediately frozen in liquid nitrogen and stored at ultra low temperature (-70oC) for further analysis of the transcriptome.
|
| Growth protocol |
The fed-batch fermentations with yeast cell recycle were conducted in bench scale reproducing all steps that occur in a typical industrial process of alcoholic fermentation in Brazil. Fermentations were carried out in triplicate for each strain. After centrifugation, the yeast cells were resuspended in water and fed with sugar cane must containing 18% sugars, being 50% from molasses and 50% from sugar cane juice. Fermentations were conducted at 33oC for 9 hours containing 10% of yeast cells (w / v). At the end of the first cycle, fermentative yeasts were centrifuged again and the cream of yeast treated with sulfuric acid to reach pH 2.5 for 1.5 hours at room temperature. After the acid treatment, yeast cells were fed with sugar cane must to start a new fermentation. In the second cycle, fermentation samples were collected for analysis of the transcriptome.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol following manufacturer's instructions, followed by DNAse treatment and purification using the RNeasy Mini Kit (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
Prior to labelling, cDNA was synthesized from 5 µg of RNA using Agilent's cDNA Master Mix. cRNA amplification and labeling were performed by adding to the samples the Agilent Transcription Master Mix plus Cyanine-3 or Cyanine-5 for 2 hours at 40 oC.
|
| |
|
| |
| Hybridization protocol |
For the hybridization, 825 ng of each labeled cRNA was mixed with Agilent Fragmentation Mix and incubated at 60 °C for exactly 30 minutes to fragment RNA. The fragmentation was interrupted by adding 55 µL of 2X GE Hybridization Buffer HI-RPM. Finally, 100 µL of sample was placed down onto the microarray slide, which was mounted into the Agilent Microarray Hybridization Chamber Kit. The hybridization was carried out in an oven (Agilent G2545A Hybridization Oven) set to 65 °C for 17 hours. After, microarray slides were washed according to Agilent’s instructions.
|
| Scan protocol |
Scanned using the GenePix 4000B microarray scanner (Molecular Devices, USA).
|
| Description |
251632210139_GE2-v5_95_Feb07_1_4
Dye-swap of biological replicate 2.
|
| Data processing |
Agilent Feature Extraction Software (v 9.5.3.1) was used for background subtraction and LOWESS normalization. After, data were processed using the TIGR platform. Data were visualized using TMeV from TIGR, and the same software was used for statistical analysis (t test).
|
| |
|
| Submission date |
Jan 13, 2011 |
| Last update date |
Jan 14, 2011 |
| Contact name |
Patricia Alves de Castro |
| Organization name |
USP
|
| Department |
Pharmaceuticals Sciences
|
| Lab |
Molecular Biology
|
| Street address |
Av. do Café
|
| City |
Ribeirão Preto |
| State/province |
São Paulo |
| ZIP/Postal code |
14.040.903 |
| Country |
Brazil |
| |
|
| Platform ID |
GPL9825 |
| Series (2) |
| GSE26618 |
Saccharomyces cerevisiae bottom of the fermentor vs. fermentation |
| GSE26619 |
Saccharomyces cerevisiae bottom of the fermentor vs. feeding and fermentation |
|
