BioSample

Protocols: Samples for RNA isolation were taken during exponential growth phase (OD ~2) by rapid centrifugation (~30 s, max. speed) in 1.5 mL reaction tubes and quenching of cell pellets in liquid nitrogen. Pellets were stored at -80°C prior to RNA isolation. Three single colonies of C. necator H16 grown on solid LB media were spread on solid minimal media containing fructose (25 mM) and 3-HP (50 mM) as sole carbon source, respectively. Inoculated minimal media plates were incubated for two days at 30 °C. Precultures were prepared in 50 ml PE tubes containing 10 mL minimal media. They were inoculated with single C. necator colonies grown on the same carbon source and incubated at 30°C and 180 rpm in a shaking incubator. Precultures with fructose as sole carbon source were incubated for 1 day, whereas precultures with 3-HP were incubated for 2 days. Main cultures were performed in 25 mL of the same minimal media in 250 mL baffled shake flasks and were inoculated from precultures to a starting OD600 of 0.025. They were incubated at 30 °C and 180 rpm in a shaking incubator and growth was monitored by measuring optical density at 600 nm. Cell pellets were disrupted using the FastRNA PRO BLUE KIT (MP Biomedicals). On ice, two cell pellets of each culture were resuspended in 500 µL of RNApro™ solution (containing Trizol) and combined in a single 2 mL screw-cap tube with Lysing Matrix B. Cell pellets were disrupted in a tissue homogenizer (Precellys 24, Peglab) at 6000 rpm for 40 seconds. After centrifugation at 12.000 x g the supernatant was transferred to a new microcentrifuge tube and incubated at RT for 5 min, before RNA was extracted from the lysate using a DirectZol RNA Miniprep kit (Zymo Research), according to the manufacturer’s protocol. After elution of RNA in a total volume of 50 µL RNAse-free water, the samples were treated with 2.6 U of Ambion TURBO DNAse in a total reaction volume of 60 µL for 1 h at room temperature. The RNA was purified again using the Clean and concentrator kit (Zymo Research) according to the manufacturer instructions. The absence of DNA in the total RNA preparation was confirmed by PCR using two individual primer pairs and genomic C. necator H16 DNA as a positive control. 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, Biblingen, Germany TruSeq Stranded mRNA protocol ILLUMINA PROPRIETARY Part # 15031047 Rev. DSample Preparation Guide