|
| Status |
Public on Aug 28, 2012 |
| Title |
C. tropicalis Opaque Block 7 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
C. tropicalis opaque cells
|
| Organism |
Candida tropicalis |
| Characteristics |
strain: CAY2275
colony and cellular morphology: opaque state
|
| Biomaterial provider |
Bennett Lab
|
| Growth protocol |
Cells grown overnight in liquid Spider media, diluted to 0.2 OD600, and grown to 1. 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
|
| Organism |
Candida tropicalis |
| Characteristics |
reference: White and Opaque isolates of CAY1504 (white) and CAY2275 (opaque), RNA samples mixed
|
| Biomaterial provider |
Bennett Lab
|
| Growth protocol |
Cells grown overnight in liquid Spider media, diluted to 0.2 OD600, and grown to 1. Cells spun down and flash frozen in an ethanol/dry ice bath.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extracted using Ribopure Yeast Kit. RNA from 8 samples combined to comprise universal reference sample.
|
| 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.
|
| Data processing |
Data normalized using Goulphar. Mnorm value converted from log2 change by using 2^n of Mnorm Value, which yields the value in the PRE_VALUE column.
|
| |
|
| Submission date |
Aug 16, 2012 |
| Last update date |
Aug 28, 2012 |
| 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) |
| GSE40179 |
Discovery of a phenotypic switch regulating sexual mating in the opportunistic fungal pathogen Candida tropicalis |
|
