|
| Status |
Public on Nov 06, 2015 |
| Title |
S. cerevisiae_evolved_d11_empty vector_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
evolved lineage d11, non-mistranslating, replicate 2
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
evolution step: evolved
plasmid: empty
|
| Treatment protocol |
A 20-week-long laboratory experimental evolution (SC-leucine) was carried in lineages B3, D11 and H9. 1ul of stationary phase cultures were transferred to fresh media every second days using handheld pintools.
|
| Growth protocol |
Ancestor and evolved lineages grew in liquid media (SC-leucine)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the hot-phenol method. Total RNA samples were treated with DNaseI (Amersham Biosciences) according to the commercial enzyme protocol and quantification and quality control was performed using the Agilent 2100 Bioanalyzer system.
|
| Label |
Cy3
|
| Label protocol |
Labeling was performed using the Agilent protocol for One-Color MicroarrayBased Gene Expression Analysis Quick Amp Labeling v5.7 (Agilent Technologies). cDNA was synthesized from 600 ng of total RNA using Agilent T7 Promoter Primer and T7 RNA Polymerase Blend and labeled with Cyanine 3-CTP. Labeled cDNA was purified with RNAeasy mini spin columns (QIAGEN) to remove residual Cyanine 3-CTP. Dye incorporation and quantification was monitored using the Nanodrop 1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
To prepare hybridization, 1.65 μg of Cy3-labeled cRNAs were mixed with the fragmentation mix (Blocking Agent and Fragmentation Buffer) and incubation lasted for 30 minutes at 60 ºC. Finally, GEx Hybridization Buffer HI-RPM was added and the preparation was assembled in the custom made Agilent arrays (yeast G4813A). Slides were prepared using Agilent gasket slides according to the manufacturer instructions. Each hybridization was carried out for 17 hours at 65ºC, in an Agilent hybridization oven.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565AA) 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 60%).
|
| Description |
gene expression of non-mistranslating yeast after lab evolution
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 016322_D_F_20130122) to obtain background subtracted and spatially detrended Processed Signal intensities.
|
| |
|
| Submission date |
Feb 06, 2015 |
| Last update date |
Nov 06, 2015 |
| Contact name |
Ana Rita Bezerra |
| E-mail(s) |
armbezerra@ua.pt
|
| Organization name |
University of Aveiro
|
| Department |
Health Sciences
|
| Lab |
iBiMED
|
| Street address |
Campus Universitário Santiago
|
| City |
Aveiro |
| ZIP/Postal code |
3810-193 |
| Country |
Portugal |
| |
|
| Platform ID |
GPL9825 |
| Series (1) |
| GSE65718 |
Evolution of robustness to protein mistranslation by accelerated protein turnover |
|
