title: DNA-seq of Clostridium tyrobutyricum
design: Bacterial stocks were streaked onto reinforced clostridial agar (RCA) plates (Merck, Darmstadt, Germany) and incubated for 72 h at 37 C under anaerobic conditions using a jar gassing system and a gas mixture containing 80% N2, 10% CO2, and 10% H2 (Don Whitley Scientific, West Yorkshire, UK). Species assignment of the strains was conducted based on 16S rDNA sequencing according to the procedure described by Brändle et al. 2018 and confirmed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) using a Microflex LT instrument and MALDI Biotyper software (Bruker Daltonik, Bremen, Germany) according to the manufacturer's instructions. Single colonies were inoculated into reinforced clostridial medium (RCM: 10 g meat extract, 10 g tryptone, 3 g yeast extract, 10 g D(+)-glucose anhydrous, 1 g starch, 5 g sodium chloride, 3 g sodium acetate, and 0.5 g L-cysteine hydrochloride monohydrate per liter) and incubated for 72 h at 37 °C under anaerobic conditions (as described above). Then, 1.2 ml of overnight culture was pelleted at 8,000 × g for 10 min at 4°C. Genomic DNA was extracted using peqGOLD Bacterial DNA Kit (Peqlab, Erlangen, Germany) according to the manufacturer's instruction except for the following modifications: the initial lysis step using lysozyme was performed at 37 °C for 30 min. In the elution step, the DNA was eluted with 30 µl elution buffer and incubated for 5 min at 70 °C without agitation in an Eppendorf thermomixer. To increase the DNA yield, an additional elution step was performed using the first eluate. The quality was checked on 1% agarose gel run at 80 V, 2.000 mA and 300 Watt for 90 min, and the concentrations were determined using Qubit double-stranded DNA HR assay kit and Qubit fluorometer (Life Technologies, Grand Island, NY) according to each manufacturer's instructions. Libraries were prepared using the NEBNext DNA Library Prep Kit (New England, BioLabs, USA). To check the prepared DNA libraries, Qubit 2.0 fluorometer was firstly used to determine the concentration of the library. After dilution to 1 ng/l, the Agilent 2100 bioanalyzer was used to assess the insert size. Afterwards, qPCR was performed to detect the effective concentration of each library. Sequencing was performed on the Illumina NovaSeq 6000 (150-bp paired-end reads) system (Illumina, USA).