|
| Status |
Public on Feb 18, 2010 |
| Title |
SEG13 |
| Sample type |
RNA |
| |
|
| Source name |
yeast cell grown to mid-log phase (O.D. = 0.6)
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: YJM789 X S96
treatment: 5uM alpha factor
treatment time: 30 min
|
| Treatment protocol |
Incubation at 30C for 30 minutes with 200rpm swirling, in the presence of 5uM alpha factor.
|
| Growth protocol |
Yeast cells were grown in 500ml YPAD medium to mid-log phase (O.D. = 0.6).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Ambion Ribopure-yeast kit was used to extract total RNA from 10 ml pheromone-treated yeast cultures, or untreated cultures as control. The RNA concentration was measured using a NanoDrop system. RNA integrity was examined by Agilent Bioanalyzer, and all samples had 260/280 ratios around 2.2, and 260/230 ratios larger than 2.0.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 1 ug RNA using the Amino Allyl MessageAmp™ II aRNA Amplification Kit (Ambion) according to the manufacturer's instructions. Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
0.8 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 250 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Yeast Gene Expression 8x15K Microarrays (design ID: 016322) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 8x15k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
SEG13
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters to obtain background-subtracted and spatially detrended Processed Signal intensities.
|
| |
|
| Submission date |
Jan 05, 2010 |
| Last update date |
Feb 17, 2010 |
| Contact name |
Wei Zheng |
| E-mail(s) |
wei.zheng@yale.edu
|
| Organization name |
Yale University
|
| Street address |
Suite 503, 300 George Street
|
| City |
New Haven |
| State/province |
CT |
| ZIP/Postal code |
06510 |
| Country |
USA |
| |
|
| Platform ID |
GPL9825 |
| Series (2) |
| GSE19634 |
Gene Expression of MATa yeast segregants (YJM789 X S96) after alpha factor treatment |
| GSE19636 |
Genetic Analysis of Variation in Transcription Factor Binding in Yeast |
|
