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| Status |
Public on Jul 25, 2017 |
| Title |
Hybrid_Saccharomyces_cerevisiae_Hyb-8_replicate 3 |
| Sample type |
RNA |
| |
|
| Source name |
Hybrid_Saccharomyces_cerevisiae_Hyb-8
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: Hyb-8
strain info: hybrid strain
genotype/variation: PHO13 deleted
|
| Growth protocol |
Yeast strains were precultured in 5 mL YPD medium containing 50 μg/mL clonNAT, 200 μg/mL zeocin, and 300 μg/mL G418 at 150 rpm and 30°C for 24 h, followed by aerobic culturing in 500 mL YPD medium with 50 μg/mL clonNAT at 150 rpm and 30°C for 48 h. The cells were then harvested by centrifugation at 3000 ×g for 10 min and washed twice with distilled water. Cells were inoculated in 50 mL YPX medium (10 g/L yeast extract, 20 g/L Bacto-peptone, and 50 g/L xylose) with 20 mM acetic acid and 15 mM formic acid in closed 100-mL bottles equipped with a CO2 outlet at an initial cell concentration of 50 g wet cells/L. Fermentation was initiated by the addition of yeast cells into the bottle. Temperature was controlled using a water bath equipped with a magnetic stirrer. Fermentation temperatures were set to 38°C with stirring at 500 rpm. Samples obtained after 3 h of fermentation were used for RNA preparation to observe levels of gene expression.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was obtained by following the protocol provided for the Total RNA Isolation Mini Kit (Agilent Technologies, Palo Alto, CA, USA). RNA concentration and quality were measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop, Delaware, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies), respectively.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 100 ug RNA using the One-Color Low-input QuickAmp Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer, and its quality checked by Agilent 2100 Bioanalyzer.
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| |
|
| Hybridization protocol |
1.65 ug of Cy3-labelled cRNA (specific activity >9.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 ul containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ul of 2x Agilent GE hybridization buffer HI-RPM was added to the fragmentation mixture and hybridized to Agilent Yeast Microarrays for 17 hours at 65°C in a rotating hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with Wash Buffer 1 (Agilent) and 1 minute with 37°C Wash Buffer 2 (Agilent).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner using one color scan setting for 4x44k array slides (Scan Region 61x21.6 mm, Scan resolution 5um, R PMT is set to 100% and G PMT is set to 100%, XRD=0.10).
|
| Description |
Hyb-8_rep3
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.1.1.1 (Agilent) using default parameters (protocol GE1-v5_10 Apr08) and Grid: 015072_D_20060913) to obtain background subtracted and spatially detrended Processed Signal intensities.
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| |
|
| Submission date |
Jul 24, 2017 |
| Last update date |
Jan 23, 2018 |
| Contact name |
Akihiko Kondo |
| Organization name |
Kobe University
|
| Department |
Chemical Science and Engineering
|
| Street address |
1-1 Rokkodai, Nada
|
| City |
Kobe |
| ZIP/Postal code |
657-8501 |
| Country |
Japan |
| |
|
| Platform ID |
GPL9294 |
| Series (1) |
| GSE101786 |
Transcriptomic analysis of a novel Saccharomyces cerevisiae strain with improved xylose fermentation ability under heat and acid co-stress |
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