|
| Status |
Public on Feb 14, 2022 |
| Title |
H7-Log phase-Rep 1 |
| Sample type |
RNA |
| |
|
| Source name |
Oxidative stress resistant Saccharomyces cerevisiae, log. Phase, replicate 1
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
tag: CEN.PK 113.7D (MATa)
|
| Treatment protocol |
There is no treatment protocol in this study.
|
| Growth protocol |
Precultures of oxidative stress resistant Saccharomyces cerevisiae (H7) mutant strain and reference strain were prepared in yeast minimal medium (2% glucose and 0.67% yeast nitrogen base without amino acid (Difco)) by incubating at 30°C and 150 rpm for about ~ 16 hour. Precultures were inoculated into fresh yeast minimal medium to an initial OD600 of 0.1. Then, cultures were incubated at 30°C and 150 rpm. When cultures OD600 value reach the of about 1.0 (correspoinding to mid-exponential phase). Cells were harvested and total RNA extraction was done.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA of yeast cells were extracted by using RNeasy Mini Kit supplied from QIAGEN according to the manufacturer's recommendations. RNA was quantified using a NanoDrop-2000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled RNA was prepared from 0.2 µg RNA using the One-color Low Input Quick Amp Kit (Agilent) according to the manufacturer's instructions, followed by Agilent Nano-prep RNA purification (Agilent, Santa Clara, CA). Dye incorporation and cRNA yield were checked with the NanoDrop-2000 spectrophotometer.
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| |
|
| Hybridization protocol |
0.6 µg of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/µg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 µL containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 µL of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Yeast V2 Oligo Microarrays (G4813A-016322) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x15k array slides (Scan Area 61x21.6 mm, Scan resolution 5µm, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression in the log phase of cell growth
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 016322_D_F_20070321). Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
Sep 28, 2021 |
| Last update date |
Feb 15, 2022 |
| Contact name |
Halil ibrahim KISAKESEN |
| E-mail(s) |
[email protected]
|
| Phone |
+905307831229
|
| Organization name |
Istanbul Technical University
|
| Department |
Molecular Biology, Genetics and Biotechnology
|
| Lab |
Yeast
|
| Street address |
ITU MOBGAM, SARIYER
|
| City |
Istanbul |
| State/province |
Istanbul |
| ZIP/Postal code |
34469 |
| Country |
Turkey |
| |
|
| Platform ID |
GPL22543 |
| Series (1) |
| GSE184952 |
Physiological and Molecular Characterization of an Oxidative Stress-Resistant Saccharomyces cerevisiae Strain Obtained by Evolutionary Engineering |
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