|
| Status |
Public on Jan 30, 2015 |
| Title |
pap1 deletion mutant control, biological rep2 |
| Sample type |
RNA |
| |
|
| Source name |
pap1 deletion mutant_control
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: AV25
genotype/variation: pap1 deletant; h- pap1::kanMX6
treated with: decomposed detaNONOate solution (control)
|
| Treatment protocol |
Stress treatments were done for exactly 15 minutes at 30°C in flasks shaking at 140 rpm. Stress conditions are described as follows: Nitrosative stress: Pure Nitric oxide donor compound detaNONOate (Enzo Life Sciences) was dissolved in 0.01N NaOH solution to a stock solution of 100 mM and added to the cells at a final concentration of 3 mM. A decomposed detaNONOate solution was added to the cells at the same concentration as control. The control and the treated cells were harvested by gentle centrifugation of 3000 rpm for 3 minutes and cell pellets were immediately resuspended in RNAlater solution (Ambion; Life Technologies) and frozen in -80°C freezer.
|
| Growth protocol |
The two strains were cultured in yeast extract (YE) medium at 30°C, shaken in flasks at 140 rpm until reaching O.D.600 = 0.5 (1 x 10^7 cells/ml). Cells were harvested from the medium, re-suspended in 1x PBS to the same density and distributed from the same flask as sets of controls and treatment for both the strains.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated according to the protocol provided in RNeasy Mini Handbook 04/2006 from Qiagen page 45 using RNeasy Mini Kit. The lysis was done according to mechanical lysis method provided in the handbook.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 100 ng total RNA (Affymetrix 3’ IVT Express manual).
|
| |
|
| Hybridization protocol |
Following fragmentation, 5 ug of cRNA were hybridized for 16 hr at 45C at 60 rpm on Yeast 2 Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
|
| Scan protocol |
GeneChips were scanned using the GeneChip Scanner 3000 7G.
|
| Description |
AV25 C2
AV25 C2_(Yeast_2).CEL
|
| Data processing |
The raw Affymetrix data files (.cel) were pre-processed using the software RMA Express (v1.0.4), In RMA express the output data file is a .txt file (tab delimited text file) which is read for analysis in Multi Experiment Viewer (MEV). In MEV, we loaded data as RMA file under Affymetrix file section; we downloaded the annotation file for yeast 2 array from Affymetrix website. The annotation file was loaded after loading the expression data file. We set the percentage cut off filter at 100 i.e. all genes having significant expression in all the samples would only be included in analysis. Next we normalized Genes/Rows i.e. this would transform values using the mean and the standard deviation of the row of the matrix to which the value belongs, using the following formula: Value = [(Value) – Mean (Row)]/[Standard deviation (Row)]. We found out the differentially expressed genes between the control and treated sets using one way ANOVA using the threshold p-value 0.01. The genes that were differentially expressed between the 972h- Treated and 972h- Control by a minimum of 2 folds were considered significant. Hierarchical Clustering of the significantly induced and repressed genes were performed using the metric Pearson correlation co-efficient with the linkage method being Average Linkage.
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| |
|
| Submission date |
Sep 16, 2013 |
| Last update date |
Jan 30, 2015 |
| Contact name |
Sanjay Ghosh |
| E-mail(s) |
[email protected]
|
| Phone |
913324615445
|
| Organization name |
University of Calcutta
|
| Department |
Biochemistry
|
| Street address |
Dept. of Biochemistry, 35, Ballygunge Circular Road, Kolkata-700019
|
| City |
Kolkata |
| State/province |
West Bengal |
| ZIP/Postal code |
700019 |
| Country |
India |
| |
|
| Platform ID |
GPL2529 |
| Series (1) |
| GSE50899 |
Expression data from fission yeast Schizosaccharomyces pombe under nitrosative stress |
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