|
| Status |
Public on Feb 23, 2010 |
| Title |
Sp-CONT-Biorep1 |
| Sample type |
RNA |
| |
|
| Source name |
Wildtype S.pombe strain
|
| Organism |
Schizosaccharomyces pombe 972h- |
| Characteristics |
oxidative stress: no
|
| 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 was performed following the instructions of the GeneChip whole transcript double-stranded target-labelling assay Affymetrix manual.
|
| |
|
| Hybridization protocol |
Hybridization was performed using 5 micrograms of cRNA, 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 GPL2529-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 |
GPL9671 |
| Series (1) |
| GSE19020 |
Analysis of DNA strand-specific differential expression with high density tiling microarrays |
|
