Computational protocol: The Anaerobically Induced sRNA PaiI Affects Denitrification in Pseudomonas aeruginosa PA14

Similar protocols

Protocol publication

[…] Total RNA was prepared from three biological replicates of PA14 and PA14ΔpaiI, respectively. The overnight cultures were inoculated in fresh BSM medium supplemented with 20 mM glucose at an initial OD600 of 0.05. PA14 and PA14ΔpaiI were first grown aerobically to an OD600 of 0.4, at which KNO3 (final concentration 100 mM) was added and then transferred to an anaerobic chamber. After 1 h of incubation, the cells were harvested for total RNA preparation using the TRIzol Reagent (Ambion) according to the manufacturer's instructions. Under these conditions anaerobiosis was ensured by employing an anaerobic indicator strip (Oxoid). The samples were DNase I treated, followed by phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation. The Ribo-Zero™ rRNA Removal Kit (Illumina) was used to deplete rRNA from total RNA samples. Libraries were constructed using the NEBNext1Ultra™ Directional RNA Library Prep Kit from Illumina. 100 bp single end sequence reads were generated using the Illumina HiSeq 2000 platform at the Vienna Biocenter Core Facilities (http://www.csf.ac.at). The sequence reads were adaptor clipped and quality trimmed with trimmomatic (Bolger et al., ) with default parameters. Mapping of the samples against the UCBPP-PA14 reference genome (accession number NC_008463) was performed with Segemehl (Hoffmann et al., , ). The uniquely mapped sequencing data were split by strand and further processed for automatic UCSC Genome Browser visualization with the ViennaNGS toolbox (Kent et al., ; Wolfinger et al., ). Read counting for subsequent DESeq2 (Love et al., ) differential gene expression analysis was performed with BEDtools (Quinlan and Hall, ). The raw sequencing reads were deposited in the European Nucleotide Archive as a study under the accession number PRJEB22593 (http://www.ebi.ac.uk/ena/data/view/PRJEB22593). […]

Pipeline specifications

Software tools Trimmomatic, Segemehl, ViennaNGS, DESeq2, BEDTools
Databases ENA UCSC Genome Browser
Application RNA-seq analysis
Diseases Cystic Fibrosis, Neoplasms
Chemicals Carbon, Glucose, Nitrates, Nitric Oxide, Nitrites