Project description:Oligotropha carboxidovorans strain OM5 is an aerobic carboxidotrophic bacterium and potentially a promising candidate for such processes. We here performed RNA-Seq analysis comparing cells of this organism grown heterotrophically with acetate or autotrophically with CO2, CO and H2 as carbon and energy source and found a variety of chromosomally and of native plasmid-encoded genes to be highly differentially expressed. For RNA extraction and RNA-Seq, respectively, six aliquots of the sample of each culture replicate were pooled to gain enough cell mass and RNA, accordingly, while four biological replicates were processed for the RNA-Seq analysis. Total RNA was isolated from four biological replicates using Zymo Quick-RNA Miniprep Plus kit with bead beating and DNase-treatment (Zymo Research, Freiburg, Germany). Initially RNA quality was checked by Trinean Xpose (Gentbrugge,Belgium) and Agilent RNA Nano 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). Samples contaminated with DNA were treated with DNase again, cleaned as described above and rechecked by Xpose and Agilent Bioanalyzer. Finally RNA was free of DNA with an RNA Integrity Number (RIN) > 9 and rRNA Ratio [23s / 16s] > 1.5. Ribo-Zero rRNA Removal Kit (Bacteria) from Illumina (San Diego, CA, USA) was used to remove the ribosomal RNA molecules from the isolated total RNA. Removal of rRNA was checked by Agilent RNA Pico 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). RNA was free of detectable rRNA. TruSeq Stranded mRNA Library Prep Kit from Illumina (San Diego, CA, USA) was used to prepare cDNA libraries. TruSeq stranded cDNAs were sequenced paired end on an Illumina HiSeq1500 system, rapid mode using 70 bp read length.
Project description:RNA-seq of Oligotropha carboxidovorans strain OM5, a promising candidate for the aerobic utilization of synthesis gas, heterotrophically (acetate) and autotrophically (syngas) grown.
