Amplicon sequencing of fecal samples of mice colonized with human gut bacteria

Germfree mice were inoculated with glycerol stocks of the frozen communities (human gut bacteria). Mice were inoculated by oral gavage (50 µl) and inoculation was repeated after 48 h using the same protocol. The germfree control group was left untreated. 10 days after the second inoculation with human gut bacteria, mice were orally gavaged with PFNA (10 mg/kg or 0.1 mg/kg in 25 % DMSO) in a volume of 50 µl. Fresh fecal samples were collected before treatment, 3 h, 1 day and 2 days after treatment in sterile 1.5 ml Eppendorf-tubes and immediately frozen at -80 °C. On day 3 after treatment, mice were euthanized by CO2 and cervical dislocation, dissected and intestinal contents were taken from colon and the small intestine and collected in the same way. DNA extraction from fecal samples was performed in house using MagMAXTM Microbiome Ultra Nucleic Acid Isolation Kit in bead tubes (Thermo Fisher Scientific, A42358) and the KingFisherTM Flex (MAN0018071) according to the manufacturer's instructions. After, DNA integrity was verified using agarose gel electrophoresis and the DNA concentration was determined using QubitTM dsDNA BR assay kit (Thermo Fisher Scientific, Q32853) in combination with the Varioskan LUX plate reader (thermoscientific).