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RNA Seq of passages of a small colony variant P. aeruginosa clinical isolate

ABSTRACT: Purpose : The goal of this study was to use RNA Seq to explore the correlation of gene expression of a collection of clinical P. aeruginosa isolates to various phenotypes, such as colony morphology, c-di-GMP production and biofilm formation Methods : mRNA profiles were generated for Pseudomonas aerugionsa clinical samples by deep sequencing.Ribosomal RNA was removed by using the RiboZero Bacteria kit (Illumina). cDNA libraries were synthesized using the SMARTScribe Reverse Transcriptase (Takara) followed by a PCR enrichment using the AccuPrime HiFi Taq polymerase (Invitrogen). Enzymatic reactions were carried out in the presence of SUPERase·In™ RNase Inhibitor (Invitrogen); RNACleanXP beads (Agencourt) were used for all RNA purification steps. Quality checks were performed before, during and after cDNA library preparation with the RNA Nano Kit and an Agilent Bioanalyzer 2100 (Agilent Technologies). Libraries were sequenced on an Illumina NovaSeq 6000 (paired-end mode; 2 x 50 bp) and mRNA reads were trimmed using the tool ‘cutadapt’ (version 3.5) with customized settings and mapped to the NC_008463.1 (PA14) reference genome from NCBI using ‘bowtie2’ (version 2.3.5.1) with the settings “--very-sensitive-local; --no-mixed; --fr; --no-unal”.

ORGANISM(S): Pseudomonas aeruginosa

PROVIDER: GSE147921 | GEO | 2022/11/05

REPOSITORIES: GEO

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Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare adhesion gene clusters and clock genes / biomarkers at different time points throughout the day in sorted murine lympahtic endothelial cells. Methods: RNA of sorted dermal LECs harvested in TriZol-LS was purified using Direct-zol RNA mini Prep Kit (ZymoResearch) following manufacturer’s instructions. Isolated RNA was quantified using the Nanodrop (Thermofisher) and analyzed on a Bioanalyzer (Agilent) using the RNA 6000 Nano or Pico Kit (Agilent). 75 ng of eluted total RNA was digested with DNase (Thermofisher) to remove DNA contaminations. An additional purification step with RNAClean XP Beads (Agencourt) was performed. The purified, bead-bound RNA was directly used as input in the SMART-Seq v4 Ultra Low Input RNA Kit (TaKaRA Bio). Full-length cDNA was generated following manufacturer’s instructions. Full-length cDNA was quantified using the Qubit dsDNA HS Assay Kit (Invitrogen) with the Qubit fluorometer. Finally, sequencing libraries were generated using 500 pg full-length cDNA following the NexteraXT protocol (Illumina). Libraries were quantified on a Bioanalyzer (Agilent) using the DNA 1000 Kit (Agilent) and sequenced on a HiSeq1500 system (Illumina) with a readlength of 100 nt, single-end mode. Results: obtained transcriptome profiles were processed on a Galaxy web interface hosted by LAFUGA (Gene Center, Munich). After demultiplexing and trimming data was mapped against the mouse genome (mm10) using RNA-STAR mapper (Galaxy Version 2.5.2b-0). Abundant reads were counted using HTSeq-count (Galaxy Version 1.0.0). Afterwards, gene expression analysis to detect differentially expressed genes was performed using DESeq2 (Galaxy Version 2.11.40.6), setting the false discovery rate (FDR) <0.05. Comparing the expression profiles and the enrichment of gene ontology cluster adhesion revealed a highly time-of-day dependent expression profile of genes. Conclusions: Our study represents the first detailed analysis of time-of-day dependent lymphatic endothelial cell transcriptomes, with respect to adhesion and clock genes. This will provide valid input into chronotherapy, timed delivery of substances and new therapeutic avenues.

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RNA Seq of passages of a small colony variant P. aeruginosa clinical isolate

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