|
| Status |
Public on May 19, 2017 |
| Title |
SRM11-Log phase-Rep 2 |
| Sample type |
RNA |
| |
|
| Source name |
Long-lived Saccharomyces cerevisiae, log. Phase, replicate 2
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
background strain: CEN.PK 113.7D (MATa)
genotype/variation: SRM11
|
| Treatment protocol |
There is no treatment protocol in this study
|
| Growth protocol |
Precultures of SRM11 and reference strain were prepared in yeast minimal medium (2% glucose and 0.67% yeast nitrogen base without amino acids (Difco)), and incubated at 30°C and 150 rpm for ~ 16 h. Precultures were then inoculated in triplicate into fresh yeast minimal medium, to an initial OD600 of 0.1. Cultures were incubated at 30°C and 150 rpm. When the culture OD600 values reached 1.0, 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 (QIAGEN), according to the manufacturer's recommendations. RNA was quantified using a NanoDrop-2000 spectrophotometer and quality (RNA Integrity Number-RIN) was monitored by the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled RNA was prepared from 200 ng 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 using the NanoDrop-2000 spectrophotometer.
|
| |
|
| Hybridization protocol |
0.6 µg of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/µg cRNA) was fragmented at 60°C for 30 min in a reaction volume of 25 µL containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturer’s 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 h at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed for 1 min at room temperature with GE Wash Buffer 1 (Agilent) and 1 min with 37°C GE Wash buffer 2 (Agilent)
|
| Scan protocol |
Immediately after washing, slides were scanned 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 using Feature Extraction Software 9.1 (Agilent) with default parameters (protocol GE1-v1_91 and Grid: 016322_D_F_20070321). Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
May 18, 2017 |
| Last update date |
Jan 23, 2018 |
| Contact name |
Zeynep Petek Cakar |
| E-mail(s) |
bioibo@gmail.com
|
| Organization name |
Istanbul Technical University
|
| Department |
Molecular Biology, Genetics and Biotechnology
|
| Street address |
ITU MOBGAM, SARIYER
|
| City |
Istanbul |
| State/province |
Istanbul |
| ZIP/Postal code |
34469 |
| Country |
Turkey |
| |
|
| Platform ID |
GPL16244 |
| Series (1) |
| GSE99041 |
Transcriptomic analysis of a Saccharomyces cerevisiae mutant with extended chronological life span |
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