|
| Status |
Public on Nov 22, 2013 |
| Title |
CAY2206 a wor1-deletion white 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
C. tropicalis white wor1-deletion cells
|
| Organism |
Candida tropicalis |
| Characteristics |
strain: CAY2206
|
| Growth protocol |
For experimental biofilm samples: Cultures were inoculated in 3 ml of Spider medium then incubated at 25°C overnight. 2 ODs of cells were spun down and resuspended in 1 ml of Lee's + Glucose medium and transferred to the well of a 12-well polystyrene plate. Plates were incubated at 25°C for 1-2 days without shaking, and cells were removed ands spun down; For universal reference samples: 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 Cy5. Samples were incubated at room temperature for 1 hour in darkness and cleaned up using Zymo columns.
|
| |
|
| Channel 2 |
| Source name |
Universal reference RNA
|
| Organism |
Candida tropicalis |
| Characteristics |
strain: Mixed white and opaque grown in liquid Spider medium
|
| Growth protocol |
For experimental biofilm samples: Cultures were inoculated in 3 ml of Spider medium then incubated at 25°C overnight. 2 ODs of cells were spun down and resuspended in 1 ml of Lee's + Glucose medium and transferred to the well of a 12-well polystyrene plate. Plates were incubated at 25°C for 1-2 days without shaking, and cells were removed ands spun down; For universal reference samples: 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 Cy5. 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 1 of 2, CAY2206 wor1 deletion a white cells
|
| Data processing |
Data normalized using Goulphar. Mnorm log2 values were averaged for each spot.
|
| |
|
| Submission date |
Nov 21, 2013 |
| Last update date |
Nov 22, 2013 |
| Contact name |
Stephen Knox Jones Jr |
| E-mail(s) |
[email protected]
|
| Organization name |
Brown University
|
| Department |
Molecular Microbiology and Imunology
|
| Lab |
RJ Bennett
|
| Street address |
171 Meeting St.
|
| City |
Providence |
| State/province |
RI |
| ZIP/Postal code |
02912 |
| Country |
USA |
| |
|
| Platform ID |
GPL15925 |
| Series (1) |
| GSE52634 |
C. tropicalis cells under in vitro biofilm-forming conditions |
|
