Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq of Gordonia rubripertincta CWB2 to uncover the styrene specific degradation pathway

ABSTRACT: Gordonia rubripertincta CWB2 was grown at 30 °C in minimal media with fructose (with and without Fe) and styrene as sole source of carbon, respectively. After 5 days of cultivation the culture was diluted 1/10 in fresh media and incubated for 24 h with the respective substrate in a set of four Erlenmeyer flasks. Prior harvesting the cells, 10 % of an ice-cold STOP-solution (10 % buffered phenol in ethanol) was added to the culture followed by centrifugation at 11,000 x g for 5 minutes at 4 °C. The supernatant was discarded and the pellet was stored until RNA isolation at -80 °C. To break up the cells, 150 µl of a 5 mg ml-1 lysozyme solution were added to the pellet, mixed and incubated at room temperature for 5 minutes. 450 µl of buffer RLT (Qiagen) and 50 mg of (0.1 mm) glass beads were added to resuspend and break the cells by repeated vortexing at 4 °C. The suspension was applied to QIAshredder column for homogenization and to remove particles from the sample. Extraction of total RNA Extraction was done by applying the RNeasy Mini Kit including on-column DNA digestion (Qiagen). Isolated RNA was stored at -80 °C and quality was controlled on the 2100 Bioanalyzer using the RNA 6000 Nano Kit (Agilent). RNA quality and quantity was again checked by an Agilent 2100 Bioanalyzer run (Agilent Technologies, Böblingen, Germany) and Trinean Xpose sytem (Gentbrugge, Belgium) prior and after rRNA depletion by Ribo-Zero rRNA Removal Kit (Bacteria) (Illumina, San Diego, CA, USA). TruSeq Stranded mRNA Library Prep Kit from (Illumina, (San Diego, CA, USA) was used to prepare the cDNA libraries to analyze the whole transcriptome. The resulting cDNAs were then sequenced paired end on an Illumina MiSeq and HiSeq 1500 system (San Diego, CA, USA) using 2 x 75 nt read length.

INSTRUMENT(S): Illumina HiSeq 1500, Illumina MiSeq

ORGANISM(S): Gordonia rubripertincta

SUBMITTER: Tobias Busche

PROVIDER: E-MTAB-6012 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Actinosynnema mirum cells were grown in 3 biological replicates and harvested in exponential growth phase either grown in terrific broth (TB) or in glucose-yeast-malt (GYM) medium to analyse the differential gene expression under different grwoth media. A preculture of A. mirum was grown in 20 mL NL148 medium in 100 mL shake flasks for 3 days at 28 °C and 180 rpm (2.5 cm shaking diameter). 1 mL of the preculture was transferred to 60 mL terrific broth (TB) or 60 mL glucose-yeast-malt (GYM) medium in 500 mL shake flasks cultivated for 20 h at 28 °C and 180 rpm (2.5 cm shaking diameter). Pre and main culture were cultivated in triplicates. 495 µL of cultures was transferred to 96 well plates and 5 µL ritonavir in a final concentration of 0.1 mg mL-1 was added. Samples were incubated at 28 °C, 200 rpm for 44 h. Samples were centrifuged at maximum speed for 30 s, the supernatant was removed, and samples were flash frozen in liquid nitrogen. Cells were mechanically homogenized in 1X DNA/RNA Shield™ using Bead Tubes Type B (Macherey-Nagel, Düren, Germany) in a Bead Tube Holder at maximum speed for 20 min. RNA isolation was performed using the Quick-RNA Miniprep Plus (ZYMO research, Freiburg, Germany). An additional in-column DNase I treatment was performed. RNA pellets were eluted in 100 µL DNase/RNase-free water. The purified RNA was quantified with a NanoDrop (Thermo Scientific™, Waltham, USA). RNA quality was checked by Trinean Xpose (Gentbrugge, Belgium) and Agilent RNA Nano 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). RNA was stored at -80 °C. Total-RNA with an RNA integrity number (RIN) > 9 was used to prepare cDNA libraries. 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. Preparation of cDNA libraries were performed according to the manufacturer’s instructions of TruSeq stranded mRNA Kit (Illumina, San Diego, USA). Subsequently, each cDNA library was sequenced on a HiSeq1500 (2 x 75nt PE rapid v2) Sequencer system (Illumina, San Diego, USA).

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RNA-seq of Gordonia rubripertincta CWB2 to uncover the styrene specific degradation pathway

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