Genomic DNA was extracted from feces according to the method described by Takahashi
et al. [35 (link)]. Briefly, frozen
fecal samples were thawed on ice, and 100 mg of each sample was suspended in a solution
containing 4 M guanidium thiocyanate, 100 mM Tris-HCl (pH 9.0), and 40 mM
ethylenediaminetetraacetic acid (EDTA). The samples were disrupted using zirconia beads in
a FastPrep FP100A instrument (MP Biomedicals, Santa Ana, CA, USA). DNA was extracted from
the bead-treated suspensions using a Magtration System 12GC and GC series MagDEA DNA 200
(Precision System Science, Matsudo, Japan). For polymerase chain reaction (PCR)
amplification of the V3-V4 region of prokaryote 16S rRNA genes, a primer set consisting of
Pro341F and Pro805R was used, as previously described [35 (link)]. Sequencing was conducted at the Bioengineering Lab Co., Ltd. (Sagamihara,
Japan). Paired-end sequencing (2 × 300 bp) was performed using the Illumina MiSeq platform
(Illumina, San Diego, CA, USA) with MiSeq Reagent Kit ver. 3 (Illumina).
et al. [35 (link)]. Briefly, frozen
fecal samples were thawed on ice, and 100 mg of each sample was suspended in a solution
containing 4 M guanidium thiocyanate, 100 mM Tris-HCl (pH 9.0), and 40 mM
ethylenediaminetetraacetic acid (EDTA). The samples were disrupted using zirconia beads in
a FastPrep FP100A instrument (MP Biomedicals, Santa Ana, CA, USA). DNA was extracted from
the bead-treated suspensions using a Magtration System 12GC and GC series MagDEA DNA 200
(Precision System Science, Matsudo, Japan). For polymerase chain reaction (PCR)
amplification of the V3-V4 region of prokaryote 16S rRNA genes, a primer set consisting of
Pro341F and Pro805R was used, as previously described [35 (link)]. Sequencing was conducted at the Bioengineering Lab Co., Ltd. (Sagamihara,
Japan). Paired-end sequencing (2 × 300 bp) was performed using the Illumina MiSeq platform
(Illumina, San Diego, CA, USA) with MiSeq Reagent Kit ver. 3 (Illumina).
