title: DNA-seq of Clostridium tyrobutyricum
design: Bacterial stocks were streaked onto reinforced clostridial agar (RCA; Merck, Darmstadt, Germany) plates and incubated for 72 h at 37 C under anaerobic conditions. To confirm the assignment to the species C. tyrobutyricum, three grown colonies were randomly selected and identified using MALDI-TOF mass spectrometry (MS), using Microflex LT instrument and MALDI Biotyper software (Bruker Daltonik, Bremen, Germany) according to the manufacturers instructions. Then, single colonies were inoculated into reinforced clostridial medium (RCM) and incubated for 72 h at 37 C under anaerobic conditions. Genomic DNA was extracted using peqGOLD Bacterial DNA Kit (Peqlab, Erlangen, Germany) according to the manufacturer's instruction. 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 (2  150-bp paired-end reads) system (Illumina, USA).