Total RNA was stabilized for purification using RNAprotect™ Bacteria Reagent and extracted using the RNeasy Mini Purification kit (QIAGEN Pty Ltd, Victoria, Australia) as per the manufacturer’s protocols. Briefly, 500μl aliquots of cells in RNAprotect™ were lysed using 5 mg/ml lysozyme (L6876, Sigma-Aldrich, MO USA). DNA was removed using the RNase-Free Dnase Set (QIAGEN) and the RNA eluted in 30μl RNase-free water. RNA concentration was measured at A260 nm, with a minimum concentration of ca. 500 ng/µl required to proceed to cDNA synthesis. The quality of the RNA and presence of residual DNA were checked by formaldehyde agarose gel electrophoresis (Ausubel et al., Current protocols in molecular biology, 2003).
Growth protocol
Cells were grown to mid-log phase (optical density (OD600) = 0.5±0.05) with shaking (200 rpm) at 35C. Cells were then inoculated into 800ml Luria Broth in a Bioreactor (CDC Atlanta, GA, USA) at a 1:100 dilution and the reactor incubated in a water bath at 35C for 3 days, with a constant stir rate of 100 rpm.
Extracted molecule
total RNA
Extraction protocol
Glass slides containing attached biofilm were washed with ice-cold 1xPBS to remove biofilm. Cells were pelleted and washed again in ice-cold 1xPBS before resuspension in RNAprotect™ Bacteria Reagent. RNA was extracted using the RNeasy Mini Purification kit (QIAGEN Pty Ltd, Victoria, Australia) as per the manufacturer’s protocols. Briefly, 500μl aliquots of cells in RNAprotect™ were lysed using 5 mg/ml lysozyme (L6876, Sigma-Aldrich, MO USA). DNA was removed using the RNase-Free Dnase Set (QIAGEN) and the RNA eluted in 30μl RNase-free water. RNA concentration was measured at A260 nm, with a minimum concentration of ca. 500 ng/µl required to proceed to cDNA synthesis. The quality of the RNA and presence of residual DNA were checked by formaldehyde agarose gel electrophoresis (Ausubel et al., Current protocols in molecular biology, 2003).
Label
GeneChip® DNA Labelling Reagent (Affymetrix)
Label protocol
cDNA was synthesised, fragmented and labelled as per the Affymetrix GeneChip Expression Analysis Technical Manual (Affymetrix Corp., CA. USA). Briefly, cDNA was synthesised by using random hexamers as primers for reverse transcription (Invitrogen Australia Pty Ltd, Victoria, Australia). The primers were annealed (70 degrees C for 10 min; 25 degress C for 10 min) to 10μg total RNA. Polyadenylated control transcripts (130 pM) were added to each sample (Affymetrix) to monitor transcriptional efficiency and array performance. Transcripts were extended with SuperScript II reverse transcriptase (Invitrogen) (25 degrees C for 10 min; 37 degrees C for 60 min; 42 degrees C for 60 min and 70 degrees C for 10 min). Residual RNA was removed by alkaline treatment followed by neutralisation and the cDNA was purified using the MinElute(TM) PCR Purification kit (QIAGEN). Purified cDNA was fragmented using DNaseI (Amersham Biosciences, NSW Australia) and the fragments 3’-end-labelled using GeneChip® DNA Labelling Reagent (Affymetrix).
Hybridization protocol
Fragmented labelled cDNA was sent to the Australian Genome Research Facility (AGRF), Melbourne, Australia, for microarray analysis, where fragmentation quality was checked using a Bioanalyser 2100 (Agilent GmbH, Waldbronn, Germany) and the NanoChip protocol (Agilent). Samples were prepared for hybridization to the Pseudomonas aeruginosa Genome Array (Affymetrix) by adding 3-7μg DNA to a probe cocktail including 1×Hybridization Buffer (100mM 2-Morpholinoethanesulfonic acid, 1mM NaCl, 20mM EDTA, 0.01% Tween-20), 0.1 mg/ml Herring Sperm DNA, 0.5 mg/ml BSA, and 7% Dimethylsulfoxide (Sigma-Aldrich). 90μl of probe cocktail was loaded into a ‘test3’ array (Affymetrix) comprising 100 housekeeping genes to check cDNA quality, followed by 130μl into the full array. The chip was hybridized by rotation at 60rpm (50 degrees C for 16h) and washed using the Pseudomonas fluidics script in the Affymetrix Fluidics Station 450.
Scan protocol
The chip was scanned using the Affymetrix GeneChip Scanner 3000 at 532 nm for excitation and 570 nm for emission. CEL and CHP files were generated for analysis using the scanner operating software, GCOS.
Description
No further information.
Data processing
Microarray data were analysed using the BIOCONDUCTOR software suite. Data normalisation used the multi-array average (RMA) method in the affy package, incorporating probe level background-correction, quantile normalisation, and the use of a linear model to extract a final expression measure for each gene on each array. The resulting expression measures were then used to determine differential expression using an empirical Bayes approach within the limma package. The false discovery rate method of Benjamini and Hochberg was controlled to reduce false positives introduced by multiple simultaneous inference.
