Rnase 1

Content samples were collected from the terminal Ileum and stored at -20°C until DNA extraction. DNA was extracted using a phenol-chloroform-based method previously described (36 (link)). In brief, 500 µL of extraction buffer (200 mM Tris Roth, Dautphetal, Germany), pH 8.0), 200 µL of 20% SDS Applichem, Darmstadt, Germany), 500 µL of phenol:chloroform:isoamyl alcohol (PCI) (24:24:1) Roth, Dautphetal, Germany) were added per sample. Lysis of bacteria was performed by mechanical disruption using a Mini-BeadBeater-96 (BioSpec, Berlin, Germany) for two times 2 min. After centrifugation, aqueous phase was passed for another phenol:chloroform:isoamyl alcohol extraction before precipitation of DNA using 500 µL isopropanol (J. T. Baker, Radnor, Pennsylvania, USA) and 0.1 volume of 3 M sodium acetate (Applichem, Darmstadt, Germany). Samples were incubated at -20°C for at least several hours or overnight and centrifuged at 4°C at maximum speed for 20 min. Resulting DNA pellet was washed, dried using a speed vacuum, and resuspended in TE Buffer (Applichem) with 100 µg/ml RNase I (Applichem, Darmstadt, Germany). Crude DNA was column purified (BioBasic Inc., Braunschweig, Germany) to remove PCR inhibitors.

Feces samples were collected and stored at– 20°C until processing for DNA based 16S rRNA gene sequencing. DNA was extracted using a phenol-chloroform-based method previously described [42 (link)]. In brief, 500 μl of extraction buffer (200 mM Tris (Roth), 20 mM EDTA (Roth), 200 mM NaCl (Roth), pH 8.0), 200 μl of 20% SDS (AppliChem), 500 μl of phenol:chloroform:isoamyl alcohol (PCI) (24:24:1) (Roth) and 100 μl of zirconia/silica beads (0.1 mm diameter) (Roth) were added per feces sample. Lysis of bacteria was performed by mechanical disruption using a Mini-BeadBeater-96 (BioSpec) for two times 2 min. After centrifugation, aqueous phase was passed for another phenol:chloroform:isoamyl alcohol extraction before precipitation of DNA using 500 μl isopropanol (J.T. Baker) and 0.1 volume of 3 M sodium acetate (Applichem). Samples were incubated at—20°C for at least several hours or overnight and centrifuged at 4°C at maximum speed for 20 min. Resulting DNA pellet was washed, dried using a speed vacuum and resuspended in TE Buffer (Applichem) with 100 μg/ml RNase I (Applichem). Crude DNA was column purified (BioBasic Inc.) to remove PCR inhibitors.