|
| Status |
Public on May 14, 2010 |
| Title |
rim15 60min [100 nM rapamycin] |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
rim15Δ untreated
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: rim15Δ
treatment: Immediately before rapamycin addition
|
| Growth protocol |
All strains were freshly streaked onto YEPD plates from frozen stock at -80C before the experiment. Strains were inoculated to SC 2% glucose medium and grown to saturation overnight. The strains were then diluted next day in fresh prewarmed SC 2% glucose medium to a OD600 of 0.05. Strains were grown in water bath at 30C with shaking 160rpm and allowed to go through at least 2 generations to OD of ~0.3. Rapamycin (reconstituted to 1mg/ml in DMSO) was added to the culture (100 nM final concentration), and 5ml samples were collected at indicated timepoints via rapid vacuum filtration then immediately frozen in liquid nitrogen, then stored in -80C. Cells for reference RNA for channel 1 was collected immediately before rapamycin addition.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Qiagen RNeasy kit following manufacturer's instructions via mechanical lysis using BeadBeater.
|
| Label |
Cy3
|
| Label protocol |
325ng of total RNA was used for Agilent Linear Amplification Protocol. The reference and sample RNA were processed in parallel and combined immediately before framentation and hybridization.
|
| |
|
| Channel 2 |
| Source name |
rim15Δ 60 minutes after rapamycin addition
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: rim15Δ
treatment: 60 minutes after rapamycin addition
|
| Growth protocol |
All strains were freshly streaked onto YEPD plates from frozen stock at -80C before the experiment. Strains were inoculated to SC 2% glucose medium and grown to saturation overnight. The strains were then diluted next day in fresh prewarmed SC 2% glucose medium to a OD600 of 0.05. Strains were grown in water bath at 30C with shaking 160rpm and allowed to go through at least 2 generations to OD of ~0.3. Rapamycin (reconstituted to 1mg/ml in DMSO) was added to the culture (100 nM final concentration), and 5ml samples were collected at indicated timepoints via rapid vacuum filtration then immediately frozen in liquid nitrogen, then stored in -80C. Cells for reference RNA for channel 1 was collected immediately before rapamycin addition.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Qiagen RNeasy kit following manufacturer's instructions via mechanical lysis using BeadBeater.
|
| Label |
Cy5
|
| Label protocol |
325ng of total RNA was used for Agilent Linear Amplification Protocol. The reference and sample RNA were processed in parallel and combined immediately before framentation and hybridization.
|
| |
|
| |
| Hybridization protocol |
500ng of fluorescence-incoporated amplified RNA per reference and sample was added with Oligoarray control targets and hybridization buffer (Agilent In Situ Hybridization Kit Plus). Combined reference and sample RNA was loaded to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. Arrays were hybridized for 17 hours at 10rpm at 65C in ozone free room. After hybridization, slides were washed and scanned.
|
| Scan protocol |
Scanned on an Agilent G2505B scanner using software ChipScan A.6.3.1
Images were quantified using Agilent Feature Extraction Software (version A.7.5.1).
|
| Description |
rim15Δ 60 minutes after addition of 100nM rapamycin
|
| Data processing |
Agilent Feature Extraction Software (A.7.5.1) was used for background subtraction and LOWESS normalization.
|
| |
|
| Submission date |
Feb 26, 2010 |
| Last update date |
Feb 26, 2010 |
| Contact name |
Soyeon Lippman |
| Organization name |
Princeton University
|
| Street address |
301 Lewis Thomas Lab
|
| City |
Princeton |
| State/province |
NJ |
| ZIP/Postal code |
08540 |
| Country |
USA |
| |
|
| Platform ID |
GPL7293 |
| Series (1) |
| GSE20539 |
Rim15 and Igo1/Igo2 are required for proper transcriptional reprogramming during entry into G0 phase in budding yeast |
|
