PCR amplification and sequencing of variable regions of the 16S rRNA gene

Bacterial DNA was extracted from isolated EVs with a genomic DNA extraction kit (Bioneer Inc., Korea) as described previously [21]. PCR amplification of bacterial 16S ribosomal RNA genes was carried out with the primer set of 16S_V3_F (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3′) and 16S_V4_R (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3′), which were specific for the V3-V4 hypervariable regions of 16S rDNA.

The libraries were prepared with PCR products in accordance with the MiSeq System guide (Illumina Inc., San Diego, CA, USA), and the prepared libraries were quantified with a QIAxpert (QIAGEN, Germany). The prepared libraries were pooled at equimolar ratios, and sequenced on a MiSeq (Illumina Inc.) in accordance with the manufacturer's recommendations.