|
| Status |
Public on May 23, 2013 |
| Title |
respiration deficient pool-yeast extract and peptone with ethanol-biological rep 3 |
| Sample type |
genomic |
| |
|
| Source name |
Respiration deficient deletion pool-9 generations yeast extract and peptone with ethanol
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
pool: homozygous gene deletion collection
growth stage: 9 generations
agent: yeast extract and peptone with ethanol
|
| Treatment protocol |
500 uM copper sulfate were incorporated into growth media in the treatment samples, 3 biological replicates each, for 9 generations of growth.
|
| Growth protocol |
Growth in yeast extract and peptone (YP) media supplemented with 2% of either glucose, ethanol, glycerol, or lactate at 30°C with vigorouse shaking. Measurements of optical density at 595 nm (OD595) to monitor growth every 15 min. Samples were harvested after precisely 9 pool generations.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted from cell pellets using the YeaStar Genomic DNA kit from Zymo Research (D2002).
|
| Label |
biotin
|
| Label protocol |
The strain-specific barcodes contained in the DNA were amplified by PCR using a set of biotinylated primers.
|
| |
|
| Hybridization protocol |
PCR reactions of up- and down-tags were mixed in equal volumes (30 ul each) and hybridized overnight to custom-built TAG4 microarrays (Affymetrix).
|
| Scan protocol |
After hybridization, arrays were stained and scanned at an emission wavelength of 560 nm using a GeneChip scanner (Affymetrix).
|
| Description |
RD.9G.3
Respiration deficient deletion pool-9 generations yeast extract and peptone with ethanol-replicate 3
|
| Data processing |
The majority of strains carry two tags that hybridize to the array, an “uptag” and a “downtag”. From each array, we extracted the fluorescence intensity values for every uptag and downtag associated with the 5050 strains in the complete homozygous deletion pool, or in the case of the respiration-deficient deletion pool, the up- and downtags associated with the 331 strains in that pool. In total, this amounted to 9937, and 675 tags, respectively. The number of tags is not exactly twice the number of strains because some strains contain only one tag. These raw fluorescence values were then log2-transformed, and quantile-normalized using the normalize.quantiles function of the preprocessCore package in R. Quantile normalization was performed on four separate groups of tags; uptags from the complete deletion pool samples, downtags from the complete deletion pool samples, uptags from the respiration deficient deletion pool samples, and downtags from the respiration deficient deletion pool samples.
|
| |
|
| Submission date |
May 22, 2013 |
| Last update date |
May 23, 2013 |
| Contact name |
Ulrich Schlecht |
| E-mail(s) |
[email protected]
|
| 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 |
GPL17030 |
| Series (1) |
| GSE47175 |
A functional screen for copper homeostasis genes identifies a pharmacologically tractable cellular system |
|
