8 protocols
ID_REF = VALUE = normalized
GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.
Data analyses from three biological replicates for each of the conditions tested were performed after normalizing and summarizing probe level measurements using Robust Multiarray Average (RMA). All microarray data analysis was performed using microarray software package Partek Genomic Suite 6.5. Only genes that fit stringent criteria (expression cutoff: 50–100% stringency; p-value ≤0.01 of one-way ANOVA data corrected by Benjamini Hochberg FDR; fold-change ≥1.4) were selected for further analysis.
Following fragmentation, 1 ug of cDNA were hybridized for 16 hr at 50°C on GeneChip P. aeruginosa Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 10 ug total RNA (Expression Analysis Protocol-Technical Manual, 2001, Affymetrix).
Total RNA was purified using the RNAeasy spin column RNeasy columns (Qiagen) according to the manufacturer’s instructions and residual DNA was eliminated by DNase treatment using RNase-free DNaseI (Thermo Scientific).
Cells were grown in 15 ml of LB at 37 °C with shaking for 16 hours. Overnight precultures were diluted to an O.D600 of 0.05 in PPGAS media and grown at 25 °C and 37 °C, respectively.
Samples were harvested at O.D600 of 1.5 and cells from 2.0 ml of each culture, was suspended in 0.5 ml of fresh PPGAS media and added to 1.0 ml of RNA Protect bacteria solution (Qiagen).