|
| Status |
Public on Apr 30, 2017 |
| Title |
Barcoded PCA pool-yeast nitrogen base with ethanol and methotrexate 12h timepoint-biological rep 2 |
| Sample type |
RNA |
| |
|
| Source name |
barcoded PCA pool-yeast nitrogen base with ethanol and methotrexate, 12h timepoint
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
pool: barcoded protein-complementation assay strains
strain/background: S288c
growth stage: 12h after transfer to ethanol
agent: yeast nitrogen base with ethanol and methotrexate
|
| Growth protocol |
Growth in minimal media (yeast nitrogen base and ammonium sulfate) supplemented with 2% dextrose. Then shifted to minimal media (yeast nitrogen base and ammonium sulfate) supplemented with 2% ethanol and methotrexate at 30 °C with vigorous shaking. Samples were harvested precisely 0h, 30min, 1h, 4h and 12 hours after shift.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from cell pellets using RiboPure RNA Purification Kit for yeast (Thermo Fisher Scientific).
|
| Label |
Biotin
|
| Label protocol |
cDNA was synthesized in 10 μl reactions containing 1 μg/μl total RNA, 12.5 ng/μl Oligo(dT)12-18 primer (Invitrogen, catalog no. 18418-012), 15 units/μl SuperScript II (Invitrogen, catalog no. 18064-014), 1 x First Strand Buffer, 10 mM DTT, and 10 mM dNTPs (Invitrogen, catalog no. 18427013). After the RNA and primers were denatured for 10 min at 70°C, the remaining reagents were added, and the reaction was incubated at 42°C for 60 min. To remove the RNA template, 2 units of RNase H were then added and the mix was incubated at 37°C for 20 min and then at 95°C for 5 min. Quality of total RNA and cRNA was monitored using RNA Nano 6000 chips processed using the 2100 BioAnalyzer (Agilent).
|
| |
|
| Hybridization protocol |
220 µl hybridization cocktail containing heat-fragmented and biotin-labeled cRNA at a concentration of 0.05 µg/µl were injected into GeneChips and incubated at 45°C on a rotator in a Hybridization Oven 640 (Affymetrix) overnight at 60 rpm. The arrays were washed and stained with a streptavidin-phycoerythrin conjugate (SAPE; Molecular Probes).
|
| Scan protocol |
After hybridization, arrays were stained and scanned at an emission wavelength of 560 nm using a GeneChip scanner (Affymetrix).
|
| Description |
Ethanol.12h.2
mRNA
Streptavidin-phycoerythrin conjugate
|
| Data processing |
CEL files containing the raw data were computed from DAT array image files using the statistical algorithm implemented in MAS 5.0 (Affymetrix). Raw data were preprocessed (background adjustment, normalization, and summarization of probe sets) by using the Robust Multiarray Analysis (RMA) package from BioConductor.
|
| |
|
| Submission date |
Aug 26, 2015 |
| Last update date |
Apr 30, 2017 |
| Contact name |
Ulrich Schlecht |
| E-mail(s) |
schlecht.ulrich@gmail.com
|
| Phone |
6502136281
|
| Organization name |
Stanford University
|
| Department |
Biochemistry
|
| Lab |
Ronald W. Davis
|
| Street address |
3165 Porter Drive
|
| City |
Palo Alto |
| ZIP/Postal code |
94304 |
| Country |
USA |
| |
|
| Platform ID |
GPL2529 |
| Series (2) |
| GSE72423 |
Systematic identification of changes in the yeast protein interaction network in response to environmental, chemical, and genetic perturbation [transcriptome data] |
| GSE72425 |
Systematic identification of changes in the yeast protein interaction network in response to environmental, chemical, and genetic perturbation |
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