|
| Status |
Public on Mar 01, 2013 |
| Title |
CAY 1511 Δ/Δwor1 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
C. tropicalis wor1 mutant cells
|
| Organism |
Candida tropicalis |
| Characteristics |
strain: CAY1511 Δ/Δwor1 (CAY4043)
|
| Biomaterial provider |
American Type Culture Collection (ATCC 34139 ST-120 his-)
|
| Growth protocol |
Cells grown overnight in liquid Spider media, diluted to 0.2 OD600, and grown to 1.0 - 1.2. Cells spun down and flash frozen in an ethanol/dry ice bath.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extracted using Ribopure Yeast Kit.
|
| Label |
Cy5
|
| Label protocol |
cDNA synthesized with Oligo(dT)20 + pdN9, with aa-dUTP/dNTPs using Superscript RT III. RNA hydrolyzed with 0.2M NaOH/0.03M EDTA and neutralized with 0.3M HCl. Samples cleaned using Zymo Columns. Sample dye-coupled in 0.1 M Na Bicarbonate, pH 9.0 and adding 1.25uL Cy3 (reference) or Cy5 (experimental). Samples were incubated at room temperature for 1 hour in darkness and cleaned up using Zymo columns.
|
| |
|
| Channel 2 |
| Source name |
Universal reference
|
| Organism |
Candida tropicalis |
| Characteristics |
strain: Mixed white, opaque, Δ/Δwor1, tdh3-wor1 reference
|
| Biomaterial provider |
American Type Culture Collection (ATCC 34139 ST-120 his-)
|
| Growth protocol |
Cells grown overnight in liquid Spider media, diluted to 0.2 OD600, and grown to 1.0 - 1.2. Cells spun down and flash frozen in an ethanol/dry ice bath.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extracted using Ribopure Yeast Kit.
|
| Label |
Cy3
|
| Label protocol |
cDNA synthesized with Oligo(dT)20 + pdN9, with aa-dUTP/dNTPs using Superscript RT III. RNA hydrolyzed with 0.2M NaOH/0.03M EDTA and neutralized with 0.3M HCl. Samples cleaned using Zymo Columns. Sample dye-coupled in 0.1 M Na Bicarbonate, pH 9.0 and adding 1.25uL Cy3 (reference) or Cy5 (experimental). Samples were incubated at room temperature for 1 hour in darkness and cleaned up using Zymo columns.
|
| |
|
| |
| Hybridization protocol |
300ng of Cy3 and Cy5 samples were mixed together in a total volume of 25uL ddH2O, heated to 95C for 3 min, and cooled to room temperature. 25uL GE hybridization buffer was added to samples, mixed, and spun down. 40uL of hybridization sample was loaded onto slide, slide sealed, and incubated at 65C rotating at 10rpm for ~17 hours. Slide washed using Agilent Wash Buffers and Drying and Stabilization Solution.
|
| Scan protocol |
Scanned using Axon 4000b scanner, pixel size = 5 um, Lines to average = 2, Focus position = 15. Scanned for count ratio = 1.
|
| Description |
Biological replicate 2 of 4, CAY1511 Δ/Δwor1 cells
|
| Data processing |
Data normalized using Goulphar. Mnorm log2 values were averaged for each spot.
|
| |
|
| Submission date |
Nov 26, 2012 |
| Last update date |
Mar 01, 2013 |
| Contact name |
Richard J Bennett |
| E-mail(s) |
[email protected]
|
| Organization name |
Brown University
|
| Department |
Molecular Microbiology and Immunology
|
| Lab |
Bennett Lab
|
| Street address |
171 Meeting St
|
| City |
Providence |
| State/province |
RI |
| ZIP/Postal code |
02912 |
| Country |
USA |
| |
|
| Platform ID |
GPL15925 |
| Series (1) |
| GSE42517 |
C. tropicalis a/alpha white, opaque, tdh3-wor, wor1 mutants |
|
