|
| Status |
Public on Jan 01, 2010 |
| Title |
Rpb1-S2A-3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Rpb1-S2A total RNA
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: "552 h- rpb1-CTD-S2A-kanR"
|
| Growth protocol |
A culture of 100ml was grown at 32°C in YES medium until mid-log phase, pelleted and snap-frozen in liquid nitrogen.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared by acidic phenol extraction at 65°C for 1 hour with brief vortexing every 10 minutes. The water phase was re-extracted with acidic phenol-chloroform-IAA (24:24:1) and the RNA precipitated. About 250 µg of RNA were purified on Qiagen RNeasy column and eluted with 30 µl H20.
|
| Label |
Cy5
|
| Label protocol |
For each sample, 500 µg of total RNA was converted into labeled cRNA with nucleotides coupled to a fluorescent dye using the Low RNA Input Linear Amplification Kit (Agilent Technologies).
|
| |
|
| Channel 2 |
| Source name |
Wild type total RNA
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: "91 h-"
|
| Growth protocol |
A culture of 100ml was grown at 32°C in YES medium until mid-log phase, pelleted and snap-frozen in liquid nitrogen.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared by acidic phenol extraction at 65°C for 1 hour with brief vortexing every 10 minutes. The water phase was re-extracted with acidic phenol-chloroform-IAA (24:24:1) and the RNA precipitated. About 250 µg of RNA were purified on Qiagen RNeasy column and eluted with 30 µl H20.
|
| Label |
Cy3
|
| Label protocol |
For each sample, 500 µg of total RNA was converted into labeled cRNA with nucleotides coupled to a fluorescent dye using the Low RNA Input Linear Amplification Kit (Agilent Technologies).
|
| |
|
| |
| Hybridization protocol |
750 ng of labeled cRNA was hybridized per sample, according to standard Agilent protocols.
|
| Scan protocol |
Microarrays were scanned using an Agilent Microarray Scanner G2565CA (Agilent Technologies)
|
| Description |
Rpb1-S2A RNA vs. wild type RNA, Replicate 3
|
| Data processing |
Spot quantification was carried out using Feature Extraction software v9.5 (Agilent Technologies). Following quantification, the microarray data was lowess-normalized and an expression ratio was calculated for each microarray feature by dividing the signal intensity of the Rpb3-S2A sample by that of the wild type control.
|
| |
|
| Submission date |
Jun 08, 2009 |
| Last update date |
Jun 11, 2009 |
| Contact name |
Harm van Bakel |
| E-mail(s) |
harm.vanbakel@mssm.edu
|
| Organization name |
Mount Sinai School of Medicine
|
| Department |
Genetics and Genomic Sciences
|
| Lab |
Bakel Lab
|
| Street address |
One Gustave L. Levy Place, Box 1498
|
| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10029 |
| Country |
USA |
| |
|
| Platform ID |
GPL8679 |
| Series (1) |
| GSE16498 |
A gene-specific requirement of RNA polymerase II CTD phosphorylation on serine 2 for sexual differentiation in fission yeast |
|
