|
| Status |
Public on Mar 17, 2017 |
| Title |
Haploids and DamP collection Biological Replicate 2 (Selenomethionine 12 uM) |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Haploid, homozygous yeast deletion collection + DamP collection (Decourty et al., Cell Rep. 2014) 10 generations with 12 uM Selenomethionine
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: Mix of haploid strains derived from BY4741 (yeast deletion collection)
treatment: Selenomethionine
dose: 12 uM
|
| Treatment protocol |
Cells in exponential growth were recovered by centrifugation, washed twice in cold water and stored at -80°C until DNA extraction
|
| Growth protocol |
Cells were thawed from -80°C aliquots and left to recover for 9 hours in synthetic complete medium (MMY medium supplemented with 50 mM MES (pH 6), 2% (w/v) glucose 40 mg/l uracil, 80 mg/l adenine, 0.1 mM methionine, 0.1 mM cysteine and 80 mg/l of the other 18 amino acids). An aliquot of the pool of strains was diluted to a DO of 0.002 in 1 liter of the same medium without (control) or with 12 or 20 µM selenomethionine. Cells from each culture were harvested when the OD at 600 nm reached 2 (after 10 generations).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted through mechanical lysis (vortexing with glass beads). PCR amplification of the barcode region was followed by a second PCR amplification with Cy3 5' tagged oligonucleotide.
|
| Label |
Cy3
|
| Label protocol |
Cy3 and Cy5 5' tagged oligonucleotides were used to amplify the barcode region with universal primers. PCR products were then mixed, precipitated in the presence of linear poly-acrylamide and prepared for hybridization
|
| |
|
| Channel 2 |
| Source name |
Haploid, homozygous yeast deletion collection + DamP collection (Decourty et al., Cell Rep. 2014) untreated control
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: Mix of haploid strains derived from BY4741 (yeast deletion collection)
treatment: untreated control
|
| Treatment protocol |
Cells in exponential growth were recovered by centrifugation, washed twice in cold water and stored at -80°C until DNA extraction
|
| Growth protocol |
Cells were thawed from -80°C aliquots and left to recover for 9 hours in synthetic complete medium (MMY medium supplemented with 50 mM MES (pH 6), 2% (w/v) glucose 40 mg/l uracil, 80 mg/l adenine, 0.1 mM methionine, 0.1 mM cysteine and 80 mg/l of the other 18 amino acids). An aliquot of the pool of strains was diluted to a DO of 0.002 in 1 liter of the same medium without (control) or with 12 or 20 µM selenomethionine. Cells from each culture were harvested when the OD at 600 nm reached 2 (after 10 generations).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted through mechanical lysis (vortexing with glass beads). PCR amplification of the barcode region was followed by a second PCR amplification with Cy3 5' tagged oligonucleotide.
|
| Label |
Cy5
|
| Label protocol |
Cy3 and Cy5 5' tagged oligonucleotides were used to amplify the barcode region with universal primers. PCR products were then mixed, precipitated in the presence of linear poly-acrylamide and prepared for hybridization
|
| |
|
| |
| Hybridization protocol |
Hybridization was done in Roche RapidHyb buffer at 24°C overnight.
|
| Scan protocol |
Genepix 4200AL and GenePix Pro v7
|
| Description |
Selenometh12uM_exp2
Biological replicate 2 of 2 (12 uM Selenomethionine)
|
| Data processing |
The scans were processed using GenePix Pro, v7. The values from the obtained gpr files were filtered to keep only values for spots with an SNR > 10. Next, we performed a loess normalization on groups of probes (UP/DAMP, UP/DELETION, DOWN/DAMP, DOWN/DELETION), followed by aggregation of values for each mutant and a second loess normalization on the aggregated data.
|
| |
|
| Submission date |
Jul 13, 2016 |
| Last update date |
Mar 19, 2017 |
| Contact name |
Cosmin Saveanu |
| Organization name |
Institut Pasteur
|
| Department |
Genomes and Genetics
|
| Lab |
Genetique des Interactions Macromoleculaires
|
| Street address |
25-28 rue du docteur Roux
|
| City |
Paris |
| ZIP/Postal code |
75015 |
| Country |
France |
| |
|
| Platform ID |
GPL18088 |
| Series (1) |
| GSE84340 |
Saccharomyces cerevisiae deletion and DAmP collections chemogenomic screen with Selenomethionine |
|
