|
| Status |
Public on Oct 30, 2012 |
| Title |
Wild type osmoresponsive gene expression_2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
wild type control
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
treatment: control
time: 15 minutes
cell type: wild type (BY4741)
|
| Treatment protocol |
not stressed with 0.4M NaCl
|
| Growth protocol |
grown to mid-log phase in YPD rich medium
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted with hot phenol (Körher K and Domdey H. Methods in Enzymology 194:398-405. 1991). Hybridizations were performed between stressed and control conditions by triplicate. Results were analyzed comparing thermo-responsive gene expression respect to the control in each individual strain.
|
| Label |
Cy5
|
| Label protocol |
RNA (500 ng) was labeled using Agilent's Low Input RNA Labeling Kit, which involves reverse transcribing the mRNA in the presence of T7-oligo-dT primer to produce cDNA and then in vitro transcribing with T7 RNA polymerase in the presence of Cy3-CTP and Cy5-CTP to produce labeled cRNA
|
| |
|
| Channel 2 |
| Source name |
wild type NaCl
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
treatment: 0.4M NaCl osmotic shock
time: 15 minutes
cell type: wild type (BY4741)
|
| Treatment protocol |
osmotic shock 15 minutes 0.4M
|
| Growth protocol |
grown to mid-log phase in YPD rich medium
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted with hot phenol (Körher K and Domdey H. Methods in Enzymology 194:398-405. 1991). Hybridizations were performed between stressed and control conditions by triplicate. Results were analyzed comparing thermo-responsive gene expression respect to the control in each individual strain.
|
| Label |
Cy3
|
| Label protocol |
RNA (500 ng) was labeled using Agilent's Low Input RNA Labeling Kit, which involves reverse transcribing the mRNA in the presence of T7-oligo-dT primer to produce cDNA and then in vitro transcribing with T7 RNA polymerase in the presence of Cy3-CTP and Cy5-CTP to produce labeled cRNA
|
| |
|
| |
| Hybridization protocol |
The labeled cRNA was hybridized to the Agilent Yeast 4x44k 60-mer oligo microarray according to the manufacturer's protocol. The arrays were washed and dried.
|
| Scan protocol |
Fluorescent images were obtained using an Agilent G2565BA scanner at 100% PMT and 5 um resolution.
Images were quantified through the GenePix 6.0 software (Axon, Molecular Devices, Sunnywale, CA) using the irregular spot finding option
|
| Description |
Biological replicate 2 of 3
BY_2_BY_0.4M_NaCl_15min_2
Wild-type strain was grown at 30ºC and stressed or not with 0.4M NaCl.
|
| Data processing |
Extracted raw data were filtered and normalized using the Limma package developed within the Bioconductor project in the R statistical programming environment. The two channels were balanced by lowess normalization, followed by scaling across chips. Differential expression analysis was performed using SAM (Significance Analysis of Microarrays, Tusher et al. 2001).
|
| |
|
| Submission date |
Oct 10, 2012 |
| Last update date |
Oct 31, 2012 |
| Contact name |
Francesc Posas |
| Organization name |
Cell Signaling Group, IRB Barcelona
|
| Street address |
Carrar Baldiri Reixac 10
|
| City |
Barcelona |
| ZIP/Postal code |
08028 |
| Country |
Spain |
| |
|
| Platform ID |
GPL7542 |
| Series (1) |
| GSE41451 |
Hog1 bypasses stress-mediated downregulation of transcription by PolII redistribution and chromatin remodeling |
|
