Illumina HiSeq 1500 paired end sequencing; RNA-Seq analysis of Bacillus methanolicus MGA3 grown with and without 5-aminovalerate to investigate the transcriptional response of Bacillus methanolicus to 5-aminovalerate.
RNA-Seq analysis of Bacillus methanolicus MGA3 grown with and without 5-aminovalerate to investigate the transcriptional response of Bacillus methanolicus to 5-aminovalerate.
CTR1_p
B. methanolicus cultures were grown in MVcM or MVcMY media containing 200 mM methanol supplemented with and without 50 mM 5AVA, respectively. Cells were harvested in the mid log phase at an OD600 of 0.6  In order to perform total RNA extraction from B. methanolicus MGA3 cells, the NucleoSpin RNA isolation Kit (Machery- Nagel, Düren, Germany) and the RNase-free DNase set (Qiagen, Hilden, Germany) were used according to the manufacturer's instructions. B. methanolicus cultures were grown in minimal MVcM media containing 200 mM methanol, 15 mM mannitol or 15 mM arabitol. Cells were harvested in the middle of the exponential growth phase at an OD600 of 1.0 followed by total RNA isolation individually for each cultivation condition. The RNA material was tested for contaminating DNA using primers PRIF and PRIR for the amplification of the proI gene and primers MRF1 and MRR1 for the amplification of the mtlR gene (Supplementary Table 1). No product was obtained for any of the tested samples (data not shown). The quality of the samples was subsequently verified by capillary gel electrophoresis (Agilent Bioanalyzer 2100 system using the Agilent RNA 6000 Pico kit; Agilent Technologies, Böblingen, Germany) and the concentration checked by DropSenseTM 16 (Trinean, Ghent, Belgium). 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).