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| Status |
Public on Apr 30, 2017 |
| Title |
Barcoded PCA pool-yeast nitrogen base with gRNA directed against RBD2-biological rep 1 |
| Sample type |
genomic |
| |
|
| Source name |
barcoded PCA pool-10 generations yeast nitrogen base with gRNA directed against RBD2-biological rep 1
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
pool: barcoded protein-complementation assay strains
strain/background: S288c
growth stage: 10 generations
agent: yeast nitrogen base with gRNA directed against RBD2
|
| Treatment protocol |
FK506, CuSO4, doxorubicin, 1181-0519, atorvastatin, hydrogen peroxide, low concentration of ammonium sulfate, NaCl, Sorbitol, Menadione, High Temperature, complete synthetic amino acid mix, methionine were incorporated into growth media in the treatment samples.
|
| Growth protocol |
Growth in minimal media (yeast nitrogen base and ammonium sulfate) supplemented with 2% of either glucose or 2% ethanol at 30 °C with vigorous shaking. Measurements of optical density at 595 nm (OD595) were used to monitor growth every 15 min. Samples were harvested after precisely 3 pool generations. CRISPRi samples were harvested after 10 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.
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| |
|
| 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 |
BPII-RBD2.1
Barcoded PCA pool-10 generations yeast nitrogen base with gRNA directed against RBD2-biological rep 1.
|
| Data processing |
All cells carry two tags that hybridize to the GenFlex tag 16k array, an uptag and a downtag. From each array, we extracted the fluorescence intensity values for every uptag and downtag associated with the 1383 strains in the bar-coded PCA pool. These raw fluorescence values were quantile-normalized and then log2-transformed using the normalize.quantiles function of the preprocessCore package in R. Quantile normalization was performed separately for uptags and downtags. Fourteen experimental conditions were measured in duplicate, and the control condition (DMSO) was measured with 6 replicates. The distance between the means (fold-change or FC) of the control condition and each experimental condition was calculated separately for uptags and downtags. A moderated t-statistic was then calculated using the R package LIMMA, and the derived p-values were further converted to q-values using Benjamini & Hochberg False Discovery Rate (FDR) correction using the p.adjust function in R. A separate q-value was calculated for uptags and downtags. PCA strains with a significant increase in their tag abundance in response to an experimental condition were identified using (q-value < 0.05 for both uptag and downtag) and FC > 0.25 (for both uptag and downtag) as cutoffs. PCA strains with a significant decrease in tag abundance were identified using (q-value < 0.05 for both uptag and downtag) and FC < -0.25 (for both uptag and downtag) as cutoffs.
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| |
|
| Submission date |
Aug 24, 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 |
GPL20845 |
| Series (2) |
| GSE72336 |
Systematic identification of changes in the yeast protein interaction network in response to environmental, chemical, and genetic perturbation [interactome data] |
| GSE72425 |
Systematic identification of changes in the yeast protein interaction network in response to environmental, chemical, and genetic perturbation |
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