|
| Status |
Public on Oct 23, 2013 |
| Title |
csn5 vs WT replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
csn5/rri1 mutant
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: csn5/rri1 deleted strain
genetic background: W303
|
| Growth protocol |
strains were grown in YPD medium at 30°C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from S. cerevisiae cell cultures at 1 OD600/mL with hot acid phenol extraction method
|
| Label |
HyPer5
|
| Label protocol |
20 μg of total RNA extracted from cell culture as described above was mixed with 2 μg of 16mers oligo dT and incubated at 70 °C for 10 min. cDNA was synthesized in a final volume of 40 μl with 25 mM each of dATP, dCTP and dGTP, 15 mM of dTTP, 10 mM of aminoallyl-dUTP, 10 mM DTT and 400U of SuperScript III reverse transcriptase (Life Technologies, Carlsbad, California) in 1× reaction buffer. The samples were incubated for 2 hours at 42°C. cDNA was labeled with Cy3 and HyPer5 Post-Labeling Reactive Dye (GE Healthcare cod. 28-9224-19, Little Chalfont, United Kingdom) according to manufacturer’s protocol.
|
| |
|
| Channel 2 |
| Source name |
WT strain W303
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: wild type
genetic background: W303
|
| Growth protocol |
strains were grown in YPD medium at 30°C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from S. cerevisiae cell cultures at 1 OD600/mL with hot acid phenol extraction method
|
| Label |
Cy3
|
| Label protocol |
20 μg of total RNA extracted from cell culture as described above was mixed with 2 μg of 16mers oligo dT and incubated at 70 °C for 10 min. cDNA was synthesized in a final volume of 40 μl with 25 mM each of dATP, dCTP and dGTP, 15 mM of dTTP, 10 mM of aminoallyl-dUTP, 10 mM DTT and 400U of SuperScript III reverse transcriptase (Life Technologies, Carlsbad, California) in 1× reaction buffer. The samples were incubated for 2 hours at 42°C. cDNA was labeled with Cy3 and HyPer5 Post-Labeling Reactive Dye (GE Healthcare cod. 28-9224-19, Little Chalfont, United Kingdom) according to manufacturer’s protocol.
|
| |
|
| |
| Hybridization protocol |
cDNA Microarray Yeast 6.4k ver. 7 purchased from UHN Microarray Centre were pre-hybridized at 42 °C for at least 45 min in a solution containing, 5× saline sodium citrate buffer (SSC), 0.1% SDS and 0.1% BSA. The labeled cDNAs (Cy3 sample and HyPer5 sample mixed) were added to an equal volume of hybridization buffer containing 50% formamide, 10× SSC, 0.2% SDS pre-heated at 70°C for 3 min. Hybridization was carried out for 16 h at 42 °C and unbound DNA was washed off using 3 steps with solutions containing: 1) 1XSSC 0.2% SDS pre-heated at 42 °C; 2) 0.1X SSC 0.2 % SDS at room temperature; 3) two times 0.1X SSC at room temperature
|
| Scan protocol |
Scanned on an PerkinElmer ScanArray Lite scanner. Images were quantified using ScanArray Express and GenePix Pro 6.1 software
|
| Description |
Biological replicate 2 of 2
|
| Data processing |
LOWESS normalized, median background intensity subtraction, with Goulphar script running on R software. We filtered the data to exclude artifacts, saturated spots, and low signal spots
|
| |
|
| Submission date |
Oct 22, 2013 |
| Last update date |
Oct 23, 2013 |
| Contact name |
Valerio Licursi |
| E-mail(s) |
valerio.licursi@uniroma1.it
|
| Organization name |
Institute of Molecular Biology and Pathology (IBPM), National Research Council (CNR)
|
| Department |
c/o Dept. Biology and Biotechnologies "C. Darwin", Sapienza University of Rome
|
| Street address |
via degli Apuli, 4
|
| City |
Rome |
| ZIP/Postal code |
00185 |
| Country |
Italy |
| |
|
| Platform ID |
GPL14755 |
| Series (1) |
| GSE51563 |
Gene expression profile of a S. cerevisiae strain deleted in CSN5/RRI1 gene (systematic name YDL216C) |
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