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).
