|
| Status |
Public on May 25, 2010 |
| Title |
Pa_C0476M_2 |
| Sample type |
RNA |
| |
|
| Source name |
P. aeruginosa strain C0476M, replicate 2
|
| Organism |
Pseudomonas aeruginosa |
| Characteristics |
strain: C0476M
isogenic_group: P3a
morphology: Mucoid
sample_time: T2
|
| Growth protocol |
Clinical isolates and the reference strains were routinely grown on Difco blood agar base (BD Sciences) supplemented with 5% sheep’s blood (Remel), brain heart infusion agar (Fisher), or Synthetic Cystic Fibrosis sputum medium (SCFM), which mimics the nutritional environment of the CF lung and was previously described (Palmer KL, et al., J. Bacteriol. 189: 8079-8087). Bacterial growth in liquid media was monitored by measuring the optical density at 600 nm (OD600) during growth at 37°C with shaking at 250 rpm.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
P. aeruginosa isolates were grown in SCFM and cells were harvested at an OD600 of 0.4-0.5. Cultures were mixed 1:1 with RNAlater (Ambion), an RNA stabilizing agent. RNA was isolated using the RNeasy mini kit (Qiagen), and cDNA was prepared for Affymetrix GeneChip microarray analysis as previously described (Schuster M, et al., J. Bacteriol. 185: 2066-2079). PCR amplification of the P. aeruginosa rplU gene was used to detect DNA contamination using the primers rplU-For (5’-CGCAGTGATTGTTACCGGTG-3’) and rplU-Rev (5’-AGGCCTGAATGCCGGTGATC-3’). To assess RNA integrity, samples were subjected to agarose gel electrophoresis.
|
| Label |
biotin
|
| Label protocol |
The cDNA synthesis products were purified and fragmented by brief incubation with DNase I, and the 3' termini of the fragmentation products were labeled with biotin-ddUTP (Schuster M, et al., J. Bacteriol. 185: 2066-2079)).
|
| |
|
| Hybridization protocol |
GeneChips were washed and stained using an Affymetrix fluidics station at the University of Iowa DNA core facility, followed by manufacturer's guideline.
|
| Scan protocol |
GeneChips were canned using an Affymetrix fluidics station at the University of Iowa DNA core facility, followed by manufacturer's guideline.
|
| Description |
0476M_C1983_2.CEL
|
| Data processing |
We preprocessed microarray .CEL files with the RMA method by using the affy package (Version 1.18.2) in R (Version 2.8.1) with default options (PM probe specific correction, quantile normalization, and expression measure by median polish).
|
| |
|
| Submission date |
May 23, 2010 |
| Last update date |
Sep 04, 2013 |
| Contact name |
Taejoon Kwon |
| E-mail(s) |
tkwon@unist.ac.kr
|
| Organization name |
Ulsan National Institute of Science and Technology
|
| Department |
Department of Biomedical Engineering
|
| Street address |
50 Unist-gil
|
| City |
Ulsan |
| ZIP/Postal code |
44919 |
| Country |
South Korea |
| |
|
| Platform ID |
GPL84 |
| Series (1) |
| GSE21966 |
Transcriptional profiling of P. aeruginosa isolated from 3 individuals with cystic fibrosis over time |
|
