|
| Status |
Public on Jan 28, 2014 |
| Title |
WT_CTR1_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
WT, over-expressing CTR1, rep2
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genetic background: BY4741
genotype: wild type over-expressing CTR1
|
| Treatment protocol |
Overnight cultures grown in SC-leu medium were diluted to an OD600 of 0.6 in fresh SC-leu medium with 0.02 mM CuCl2. Cells were grown at 30°C and 180 rpm for 7 h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction was done with glass beads using the following RNA extraction buffer: 0.1 M NaCl (DEPC treated), 10 mM EDTA (DEPC treated), 5% SDS (DEPC treated) and 50 mM Tris/HCl, pH 7.5. Next, two PCI (50:49:1 Phenol:Chloroform:Isoamylalkohol) extraction steps and one Sevag (24:1 Chloroform:Isoamylalkohol/Chloroform) extraction step was included, followed by RNA precipitation with 0.1 volume 3 M NaOAc, pH 5.2 (DEPC treated) and 3 volumes of 96% EtOH (-20 °C). RNA pellet was washed twice (75% EtOH and 100% EtOH). Isolated RNA was purified with the RNeasy MinElute Cleanup Kit (QIAGEN). RNA quantification was done using the NanoDrop Spectrophotometer ND-100 (NanoDrop Technologies, Inc., Wilmington, DE) and RNA integrity was verified by BioAnalyzer run (Agilent RNA 6000 Nano Kit, Agilent 2100 Bioanalyzer).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.4 ug RNA using the One-Color Microarray-Based Gene Expression Analysis (Quick Amp Labeling) from Agilent according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
0.6 ug of Cy3-labelled cRNA (specific activity >9.0 pmol Cy3/ug cRNA) was fragmented and hybridized to the Agilent-016322 Yeast (V2) Gene Expression 8x15K Microarray (Probe Name version) using the Agilent Gene Expression Hybridization Kit following the manufacturers instructions. Hybridisation was done 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).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent microarray confocal laser scanner (G2505B) using one color scan setting for 8x15k array slides (Scan Area 61x21.6 mm, Scan resolution 5µm, Dye channel is set to Green and Green PMT is set to 100%, XDR was 0.10).
|
| Description |
Gene expression of WT cells over-expressing CTR1 after 7 hours of culture under excess copper (0.02 mM CuCl2)
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software (10.7.1.1, Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 016322_D_F_20100113) to obtain background subtracted and spatially detrended Processed Signal intensities.
|
| |
|
| Submission date |
Feb 04, 2013 |
| Last update date |
Jan 28, 2014 |
| Contact name |
Astrid Schuller |
| E-mail(s) |
Astrid.Schuller@tu-dresden.de
|
| Organization name |
TU-Dresden
|
| Street address |
Zellescher Weg 20B
|
| City |
Dresden |
| ZIP/Postal code |
01217 |
| Country |
Germany |
| |
|
| Platform ID |
GPL16244 |
| Series (1) |
| GSE44043 |
Over-expression of CTR1 delta-300 alters element and transcription profiles in yeast |
|
