Computational protocol: In vivo Host Environment Alters Pseudomonas aeruginosa Susceptibility to Aminoglycoside Antibiotics

Similar protocols

Protocol publication

[…] Total RNA was isolated using the Trizol Reagent (Thermo Fisher Scientific, USA). The quantity and integrity were determined using a NanoDrop spectrophotometer (Thermo Scientific) and a Bioanalyzer 2100 system (Agilent). For mRNA sequencing, a Ribo-Zero rRNA Removal Kit (Illumina, San Diego, CA, USA) was used to selectively remove rRNA. Random oligonucleotides and SuperScript III were used to synthesize the first strand cDNA. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase treatment. After adenylation of the 3′ ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated to prepare for hybridization. To select cDNA fragments around 300 bp in length, the library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, CA, USA). DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 15 cycle PCR reaction. The products were purified with the AMPure XP system and quantified using the Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent). The DNA library was then sequenced on a NextSeq 500 platform (Illumina) by Shanghai Personal Biotechnology Cp. Ltd.Sequence reads were mapped onto PAO1 reference genome (NC_002516.2) using a CLC genomics Workbench 8.0 (CLC Bio-Qiagen, Aarhus, Denmark). The count data of expression values were then analyzed using a DESeq package of R/Bioconductor. The differentially expressed genes were identified by performing a negative binomial test using the DESeq software, with the cut-off fold-change larger than 2. The raw sequence reads were normalized by dividing with size factors, then Log2 (N + 1) transformed. The data have been deposited in the NCBI Short Read Archive (SRA) database with an accession number SRP100146. […]

Pipeline specifications

Software tools CLC Genomics Workbench, DESeq
Application RNA-seq analysis
Organisms Pseudomonas aeruginosa, Mus musculus
Chemicals Aminoglycosides, Oxygen, Tobramycin