|
| Status |
Public on Feb 01, 2014 |
| Title |
WT a_PDA_30m_rep 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
WT a cells, PDA, 30 minutes
|
| Organism |
Clavispora lusitaniae |
| Characteristics |
strain: RSY284
|
| Growth protocol |
Cells grown overnight in liquid YPD media, 3 OD600 of cells plated onto PDA plates or YPD at the appropriate time points. Cells spun scraped off plates and flash frozen in liquid nitrogen at the given time points.
|
| 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 |
WT a cells, YPD, 2 hours
|
| Organism |
Clavispora lusitaniae |
| Characteristics |
strain: RSY284
|
| Growth protocol |
Cells grown overnight in liquid YPD media, 3 OD600 of cells plated onto PDA plates or YPD at the appropriate time points. Cells spun scraped off plates and flash frozen in liquid nitrogen at the given time points.
|
| 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 1 of 2. C. lusitaniae WT a cells incubated on PDA for 30 minutes vs. C.lusitaniae WT a cells incubated on YPD for 2 hours
|
| Data processing |
Data normalized using Goulphar. Mnorm log2 values were averaged for each spot.
|
| |
|
| Submission date |
Oct 28, 2013 |
| Last update date |
Feb 01, 2014 |
| Contact name |
Christine Scaduto |
| E-mail(s) |
Christine_Scaduto@brown.edu
|
| Organization name |
Brown University
|
| Department |
Molecular Microbiology and Immunology
|
| Lab |
Bennett
|
| Street address |
171 Meeting Street
|
| City |
Providence |
| State/province |
RI |
| ZIP/Postal code |
02912 |
| Country |
USA |
| |
|
| Platform ID |
GPL17847 |
| Series (2) |
| GSE51790 |
Candida lusitaniae haploid nutrition |
| GSE51794 |
Transcriptional Profiling of the sexual life cycle of Candida lusitaniae. |
|
