|
| Status |
Public on Apr 01, 2012 |
| Title |
PAO66711 (delta cbrB) strain_A [BSM] |
| Sample type |
RNA |
| |
|
| Source name |
Bacterial RNA
|
| Organism |
Pseudomonas aeruginosa |
| Characteristics |
strain: PAO66711
genotype/variation: delta cbrB
medium: BSM medium amended with 40mM succinate
|
| Growth protocol |
PAO1 wild type strain and PAO6673 (delta crc), PAO66711 (delta cbrB), PAO6679 (delta crcZ) strains were grown in BSM medium amended with 40mM succinate to an OD600 of 1.6 Total RNA treated with RNA protect bacteria reagent® (Qiagen) was purified.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA purification was performed by the hot phenol method as described (Leoni et al, 1996). Briefly, the cell pellets of 10 ml culture were mixed with pre-heated (65°C) 2.5 ml lysis buffer (2% sodium dodecyl sulfate, 30 mM Na-acetate and 3 mM EDTA, pH 5.5) and 5 ml phenol (pH 5.5). The mixture was vigorously shaken for 8 min and incubated for 5 min on ice and then centrifuged at 4°C for 15 min at 5000 rpm. Total RNA was obtained by phenol chloroform extraction and precipitated with ethanol and Na-acetate (pH 5.5). To eliminate DNA contamination the total RNA was treated with 40 U of DNAse I (Roche) followed by phenol-chloroform extraction and ethanol precipitation. The quality of the RNA was determined on MOPS-formaldehyde agarose-gels (Sambrook and Russel, 2001).
|
| Label |
biotin
|
| Label protocol |
The synthesis of cRNA was performed using the GeneChip® One-Cycle cDNA Synthesis according to the manufacturer' protocol (Affymetrix, Santa Clara, CA, USA). Labeling, hybridization and scanning of the samples were performed as described by Affymetrix (www.affymetrix.com).
|
| |
|
| Hybridization protocol |
Affymetrix P. aeruginosa GeneChip arrays were hybridized with 11 microgramme of labeled, amplified cRNA , washed, stained and scanned according to the protocol described in Affymetrix GeneChip® Expression Analysis Manual (Fluidics protocol EukGeWS2v5_450)
|
| Scan protocol |
Scanning was done on an Affymetrix GeneChip Scanner 7G
|
| Description |
NA
|
| Data processing |
Normalized expression signals were calculated from Affymetrix CEL files using RMA (Irizarry et al., 2003) for each tissues and each time points separately.
|
| |
|
| Submission date |
Oct 26, 2011 |
| Last update date |
Apr 01, 2012 |
| Contact name |
Sylvain Pradervand |
| E-mail(s) |
Sylvain.Pradervand@unil.ch
|
| Phone |
+41 21 692 39 08
|
| Organization name |
UNI Lausanne
|
| Department |
CIG
|
| Lab |
DNA Array Facility
|
| Street address |
Genopode
|
| City |
Lausanne |
| ZIP/Postal code |
1015 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL84 |
| Series (2) |
| GSE33241 |
Novel targets of the CbrAB/Crc carbon catabolite control system revealed by transcript abundance in Pseudomonas aeruginosa [BSM] |
| GSE33245 |
Novel targets of the CbrAB/Crc carbon catabolite control system revealed by transcript abundance in Pseudomonas aeruginosa. |
|
