Mouse feces (4 mice/group) were collected at weeks 2 and 5 by means of fecal disimpaction. The collected fecal samples were immediately mixed with an appropriate volume of RNAprotect Bacteria Reagent (Qiagen, Tokyo Japan) and stored at –80°C until analysis. Bacterial genomic DNAs were extracted as follows. After thawing, the fecal samples were subjected to centrifugation at 8000 x g for 10 min. The precipitate was suspended in 200 μl of 10 mM Tris-HCl/1 mM EDTA (TE) buffer, pH 7.0, containing 15 mg/mL lysozyme (Sigma-Aldrich, St. Louis, MO, USA). After incubation of the mixture at 37°C for 1 h, 200 μl of achromopeptidase (4000 units/mL, FUJIFILM Wako Pure Chemical) were added, and the mixture was further incubated at 37°C for 30 min. Subsequently, 400 μl of a mixture containing 2 mg/mL of proteinase K and 2% sodium dodecyl sulfate were added, and the resulting mixture was incubated at 55°C for 1 h. The mixture was then treated with phenol/chloroform/isoamyl alcohol (Life Technologies Japan, Tokyo, Japan), followed by treatment with RNase A (FUJIFILM Wako Pure Chemical), as described previously [12 (link)]. DNA was precipitated by adding an equal volume of a mixture of 20% polyethylene glycol 6000 and 2.5 M NaCl followed by centrifugation at 8000 x g at 4°C, washed with 75% ethanol, and dissolved in TE.
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