|
| Status |
Public on Nov 01, 2008 |
| Title |
Planktonic cells exp phase, biological replicate 3 |
| Sample type |
RNA |
| |
|
| Source name |
P. aeruginosa planktonic cells after 3h of growth
|
| Organism |
Pseudomonas aeruginosa |
| Characteristics |
Cultures colourless, OD~1
|
| Treatment protocol |
All cells were harvested directly into RNAlater (Ambion), kept at 5degC overnight and stored at -80degC until use.
|
| Growth protocol |
All samples were grown in AGSY medium (56 mM alanine, 17 mM K2HPO4, 86 mM NaCl, 100 μM CaCl2, 10 mM MgSO4, 5 µM FeCl2, 7.5 µM ZnCl2, 0.5% v/v glycerol, 3 g/L yeast extract, pH 7) at 37degC. Planktonic cells were grown with vigorous shaking for three hours (late exponential phase) or nine hours (early stationary phase), colonies were grown on 1.5% agar for 15 or 40 hours, and biofilms were grown for three days in silicone tubes, 6 mm i.d. under a continuous flow of 50 ml/h for 3 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the RNeasy Mini Purification Kit (Qiagen) following the supplier's instructions.
|
| Label |
biotin
|
| Label protocol |
Biotinylated RNA was prepared from 12 mg total RNA as previously described (Hentzer et al. 2005, Biofilms 2, 27-61).
|
| |
|
| Hybridization protocol |
Following fragmentation, cDNA was hybridized for 16 hr at 50degC on GeneChip P. aeruginosa Genome Arrays. The GeneChips were then washed and stained in an Affymetrix Fluidics Station 450.
|
| Scan protocol |
GeneChips were scanned using a GeneChip Scanner 3000 (Affymetrix).
|
| Description |
Gene expression data from planktonic cells in late exponential phase
|
| Data processing |
Preliminary data processing was performed using Affymetrix GCOS 1.4 and total signal target was scaled to 500 for all arrays. Normalisation was performed using
The resulting output data was analysed using R 2.5.0 (r-project.org) and the BioConductor packages affyPLM, GCRMA and LIMMA (bioconductor.org).
Normalisation was carried out using quantile normalisation, no background correction, perfect match probes only and median polish summary.
|
| |
|
| Submission date |
Jul 22, 2008 |
| Last update date |
Sep 30, 2008 |
| Contact name |
Helga Mikkelsen |
| E-mail(s) |
h.mikkelsen@imperial.ac.uk
|
| Phone |
01223 333 640
|
| Organization name |
University of Cambridge
|
| Department |
Department of Biochemistry
|
| Lab |
M. Welch lab
|
| Street address |
Building O, Downing Site
|
| City |
Cambridge |
| ZIP/Postal code |
CB2 1HA |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL84 |
| Series (1) |
| GSE12207 |
Biofilms and type III secretion are not mutually exclusive in Pseudomonas aeruginosa |
|
