Computational protocol: Transcriptome profiling reveals links between ParS/ParR, MexEF-OprN, and quorum sensing in the regulation of adaptation and virulence in Pseudomonas aeruginosa

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Protocol publication

[…] RNA-seq was performed as described previously []. Briefly, ribosomal RNA (rRNA) was depleted from ~9 μg of total RNA using the RiboZero rRNA depletion kit (for Gram-negative bacteria, Epicentre Biotechnologies, Madison, WI). Strand-specific cDNA libraries were constructed using the SOLiD Total RNA-Seq kit. Paired-end sequencing was conducted by the University of Texas Genomic Sequencing and Analysis Facility on a Life Technologies SOLiD 5500xl sequencing system with a targeted sequencing depth of six-million paired-end reads per sample. Filtering and alignment of the SOLiD 4 paired-end data was performed at the UTGSAF using the AB SOLiD BioScope Whole Transcriptome pipeline (v1.3), for whole-transcriptome RNA-seq analysis. Mapped reads were visualized using BamView in Artemis 13.2.0 [].To determine RNA transcriptional abundance for each gene, the number of reads that mapped within each annotated coding sequence (CDS) was determined. The number of reads per kb of transcript per million mapped reads (RPKM) was used to normalize the raw data [], and mean RPKM values were determined for the three biological replicates. The complete dataset including raw and processed data has been deposited at the National Center for Biotechnology Information (NCBI), Accession No. GSE44681. Comparisons were performed using a modified t-test []. A ratio of the mean RPKM values (mutant/WT) was determined for each gene. Ratios over 2 or below 0.5 and p-value < 0.05 were considered differentially expressed []. […]

Pipeline specifications

Software tools BamView, Artemis
Application RNA-seq analysis
Organisms Pseudomonas aeruginosa, Pseudomonas aeruginosa PAO1