|
| Status |
Public on Feb 23, 2010 |
| Title |
Sp-H2O2-Biorep1 |
| Sample type |
RNA |
| |
|
| Source name |
Wildtype S.pombe strain
|
| Organism |
Schizosaccharomyces pombe 972h- |
| Characteristics |
oxidative stress: yes
|
| Treatment protocol |
Two separate cultures developed from independent single colonies were processed in parallel throughout the entire experiment (biological duplicates). Cells from a 30 ml volume were collected by centrifugation at 2000 rpm for 2 minutes and the pellet was frozen immediately in liquid nitrogen (control samples). Hydrogen peroxyde (SIGMA, H-1009) was added to the rest of the culture at a final concentration of 0.5 mM and incubation proceed for 30 minutes after which 30 ml of culture were processed as above
|
| Growth protocol |
100 ml yeast extract (YE) medium at 30 ºC and 170 rpm until OD595=0.2 (4 X 106 cells/ml)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared by resuspending the cell pellets in 20 μl extraction buffer (100 mM EDTA pH 8.0, 100 mM NaCl, 50 mM Tris-Hcl pH 8.0), 20 μl phenol/chloroform, 2 μl 10% SDS, 200 μl glass beads (425-600 μm, SIGMA G-8772). Cells were mechanically disrupted in a Fast-Prep (Savant BIO 101) and the cell lysate was extracted with phenol, phenol/chloroform and chloroform/isoamyl alcohol before precipitation with 0.3 M sodium acetate and ethanol. RNA was resuspended in 50 μl of sterile water with Diethyl pyrocarbonate (SIGMA D-5758) and was furher purified through a RNeasy mini kit (Quiagen) following the suppliers specifications.
|
| Label |
biotin
|
| Label protocol |
Target labeling preserving the original polarity of RNAs was performed following the instructions of the GeneChip whole transcript sense target labelling assay manual from Affymetrix.
|
| |
|
| Hybridization protocol |
Platform hybridization was performed using 5 micrograms of single stranded sense DNA, labelled with biotin and fragmented to an average size of 100 nucleotides. Hybridization was performed following the instructions of the Affymetrix assay manual for each platform.
|
| Scan protocol |
Affymetrix standard protocol using Affymetirix Fluidics 450 and Scanner 3000
|
| Description |
Platform GPL7715-CustomCDF
|
| Data processing |
RMA
|
| |
|
| Submission date |
Nov 13, 2009 |
| Last update date |
Feb 22, 2010 |
| Contact name |
Luis Quintales |
| Organization name |
Instituto de Biología Funcional y Genómica (IBFG)
|
| Department |
Universidad de Salamanca / CSIC
|
| Street address |
Calle Zacarías González, 2
|
| City |
Salamanca |
| ZIP/Postal code |
37007 |
| Country |
Spain |
| |
|
| Platform ID |
GPL9672 |
| Series (1) |
| GSE19020 |
Analysis of DNA strand-specific differential expression with high density tiling microarrays |
|
