For biofilm growth experiments, three independent replicates of P. aeruginosa strains PAO1 and ΔmifR were grown as biofilms in a flow-through system using a once-through continuous flow tube reactor system for biofilm sample collection and in flow cells (BioSurface Technologies) for the analysis of biofilm architecture as previously described (Sauer et al., 2002, Sauer et al., 2004, Petrova & Sauer, 2009).
Extracted molecule
total RNA
Extraction protocol
Cells were treated with RNAprotect (Qiagen) and total RNA was extracted using an RNeasy mini purification kit (Qiagen) per the manufacturer’s instructions. RNA quality and the presence of residual DNA were checked on an Agilent Bioanalyzer 2100 electrophoretic system pre- and post-DNase treatment.
Label
biotin
Label protocol
Ten micrograms of total RNA was used for cDNA synthesis, fragmentation, and labeling according to the Affymetrix GeneChip P. aeruginosa genome array expression analysis protocol (end labeled with biotin-ddUTP with use of the Enzo BioArray Terminal Labeling kit (Affymetrix))
Hybridization protocol
Affymetrix GeneChip P. aeruginosa genome array expression analysis protocol.
Scan protocol
Affymetrix GeneChip P. aeruginosa genome array expression analysis protocol.
Description
SAMPLE 5
Data processing
Gene expression signal-level data were computed with the robust GeneChip multi-array average method (GCRMA) (Wu and Irizarry, 2004) using default settings, log-transformed, and normalized to total intensities with GeneSpring® GX 10.0.1 using a guided workflow. To test for differential expression between the different strains grown in a biofilm, the Bayesian adjusted t-statistics was used as implemented in the GeneSpring package. P-values were corrected for multiple testing using the Benjamini and Hochberg's method to control the false discovery rate (Benjamini and Hochberg, 1995). Only fold change ratios with adjusted P-values below 0.05 were included as statistically significant. Microarray analysis was performed on all three biological replicates. Benjamini, Y., and Hochberg, Y. (1995) Controlling the false discovery rate – a practical and powerful approach to multiple testing. J Royal Statistical Soc Series B-Methodological 57: 289–300. Wu Z, Irizarry RA: Preprocessing of oligonucleotide array data. Nat Biotechnol 2004, 22(6):656-658.
