|
| Status |
Public on Sep 01, 2011 |
| Title |
cna1_2/FK506_2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
cna1 rep 2
|
| Organism |
Nakaseomyces glabratus |
| Characteristics |
genotype/variation: cna1
|
| Treatment protocol |
Wild-type CBS138 was treated with FK506 (1 ug/ml) or CaCl2 (0.2 M)
|
| Growth protocol |
Strains were grown overnight at 24°C and washed twice by dH2O. Cells were diluted to 0.2 OD/ml in YPD and incubated 3 hr at 24°C. For wild-type, cells in log-phase were then diluted to 0.2 OD/ml (10 ml) in YPD in the presence or absence of FK506 (1 µg/ml) and CaCl2 (0.2 M). cna1 (YC98), and crz1 (YC182) mutants were diluted to 0.2 OD/ml (10 ml) in YPD. Following 3 hr incubation at 37°C/250 rpm, 10 ml of cultures were immediately poured to dry-ice/ethanol precolded 15 ml methanol (60%) to stop cellular processes and RNase activity. Cells were pelleted at 3000 rpm at -4°C, flash freezed with liquid N2, and stored at -80°C for further total RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Qiagen Yeast RNA extraction
|
| Label |
Cy5
|
| Label protocol |
Total RNA samples were reverse transcribed to cDNA and labeled with either Cy3 or Cy5 using a modification of the protocol developed by Joe Derisi (UCSF) and Rosetta Inpharmatics (Kirkland, WA) that can be obtained at www.microarrays.com.
|
| |
|
| Channel 2 |
| Source name |
FK506 rep 2
|
| Organism |
Nakaseomyces glabratus |
| Characteristics |
genotype/variation: wild-type
agent: FK506
|
| Treatment protocol |
Wild-type CBS138 was treated with FK506 (1 ug/ml) or CaCl2 (0.2 M)
|
| Growth protocol |
Strains were grown overnight at 24°C and washed twice by dH2O. Cells were diluted to 0.2 OD/ml in YPD and incubated 3 hr at 24°C. For wild-type, cells in log-phase were then diluted to 0.2 OD/ml (10 ml) in YPD in the presence or absence of FK506 (1 µg/ml) and CaCl2 (0.2 M). cna1 (YC98), and crz1 (YC182) mutants were diluted to 0.2 OD/ml (10 ml) in YPD. Following 3 hr incubation at 37°C/250 rpm, 10 ml of cultures were immediately poured to dry-ice/ethanol precolded 15 ml methanol (60%) to stop cellular processes and RNase activity. Cells were pelleted at 3000 rpm at -4°C, flash freezed with liquid N2, and stored at -80°C for further total RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Qiagen Yeast RNA extraction
|
| Label |
Cy3
|
| Label protocol |
Total RNA samples were reverse transcribed to cDNA and labeled with either Cy3 or Cy5 using a modification of the protocol developed by Joe Derisi (UCSF) and Rosetta Inpharmatics (Kirkland, WA) that can be obtained at www.microarrays.com.
|
| |
|
| |
| Hybridization protocol |
Standard Agilent protocols for Agilent Custom Oligo Microarray 8x15K G4427A for samples 5-20, and Oligo Microarray 4x44K G2519F for samples 1-4
|
| Scan protocol |
Arrays were scanned using an Agilent scanner and analyzed with Agilent’s feature extraction software version 10.5.1.1.
|
| Description |
Biological Replicate 2, wheel 2
cna1 replicate 2 vs FK506 treated replicate 2
|
| Data processing |
Agilent Feature Extraction Software (v 10.5.1.1) was used for background subtraction and LOWESS normalization. Reported expression values for each gene are median log2 ratios across all probes.
|
| |
|
| Submission date |
Aug 03, 2011 |
| Last update date |
Sep 01, 2011 |
| Contact name |
Ying-Lien Chen |
| E-mail(s) |
yc87@notes.duke.edu
|
| Phone |
9196842809
|
| Organization name |
Duke University
|
| Department |
Molecular Genetics and Microbiology
|
| Lab |
Joseph Heitman
|
| Street address |
Dept. of Molecular Genetics and Microbiology, Duke University Medical Center
|
| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27710 |
| Country |
USA |
| |
|
| Platform ID |
GPL10497 |
| Series (1) |
| GSE31167 |
Identification of calcineurin- and Crz1-dependent targets in Candida glabrata |
|
