16S rRNA Sequencing of Gut Microbiome

The fecal sample mixture was mechanically disrupted using the bead-beating method. DNA was extracted using Gene Prep Star PI-80X (Kurabo Industries, Osaka, Japan). After DNA extraction, the V3–V4 region of the 16S rRNA gene was amplified using the following primers: forward, 5′-TCG GCA GCG TCA GAT GTG TAT AAG CGA CAG CCT ACG GGN GGC WGC AG-3′; reverse, 5′-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3′ [29 (link)]. Amplicons were sequenced by the paired-end method using MiSeq (Illumina, San Diego, CA, USA). The overall procedure, from fecal sampling to 16S rRNA sequencing, was performed according to a previously described protocol [30 (link)].