|
| Status |
Public on Jul 11, 2017 |
| Title |
strainA_frozen |
| Sample type |
RNA |
| |
|
| Source name |
strainA, frozen
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: strain A
preservation: frozen
|
| Treatment protocol |
Yeast cells were precultured in YPD medium at 30°C and washed with distilled water. Wet yeast pellets were stored in the refrigerator (4°C) or in the freezer (-20°C) for two weeks. Approximately 0.4 g of yeast cells were inoculated into 30 mL of ASF medium, which consists of 23 g of sucrose, 2 g of urea, 1 g of ammonium sulfate, 0.8 g of magnesium sulfate, 1.6 mg of thiamine hydrochloride, 1.6 mg of pyridoxine hydrochloride, 16 mg of nicotinic acid in total 350 mL of 67 mM potassium phosphate buffer (pH 5.6), and incubated at 30°C. After 30 min after inoculation, yeast cells were collected, washed, and immediately frozen using liquid nitrogen.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared from whole cell lysate using the RNeasy Mini Kit (Qiagen). RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 50 ng RNA using the Low Input Quick Amp Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNeasy Mini Kit (Qiagen). Dye incorporation and cRNA yield were checked with the Agilent 2100 BioAnalyzer series II and the NanoDrop 1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
By using the Gene Expression Hybridization Kit (Agilent), 600 ng of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 24 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Technologies Yeast (v2) Gene Expression 8x15K Microarray for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with Wash Buffer 1and 1 minute at 37°C with Wash Buffer 2 of the Gene Expression Wash Buffer (Agilent), then air-dried immediately.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (Scan resolution 5 um, TIFF file dynamic change 20 bit).
|
| Description |
Gene expression during model dough fermentation of strain A cells after freezing preservation
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent). The data were imported to the GeneSpring software and normalized by 75 percentile shift.
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| |
|
| Submission date |
Jul 10, 2017 |
| Last update date |
Jan 23, 2018 |
| Contact name |
Daisuke Watanabe |
| E-mail(s) |
watanabe.daisuke@bs.naist.jp
|
| Organization name |
Nara Institute of Science and Technology
|
| Street address |
8916-5 Takayamacho
|
| City |
Ikoma |
| State/province |
Nara |
| ZIP/Postal code |
630-0192 |
| Country |
Japan |
| |
|
| Platform ID |
GPL9825 |
| Series (1) |
| GSE101071 |
Gene Expression during Model Dough Fermentation after Freezing Preservation of S. cerevisiae Baker’s Yeast Cells |
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