Sample processing and 16S rDNA sequencing
The collected EBC samples were stored in R-tubes at -20°C until sample
processing for metagenomic sequencing. Due to the relatively low sample
volume, 1x phosphate buffered saline solution (Welgene, Gyeongsan,
Korea) was added to all EBC samples to attain a total volume of 1 mL for
each sample. Subsequently, samples were centrifuged for 10 min (10,000 ×
g, 4 °C), the supernatant was collected, and passed through a 0.22-µm
filter to sterilize the solution by removing any cells or unnecessary
particles. The filtrate solution was subjected to a temperature of 100°C
for 40 min and centrifuged (13,000 rpm, 4°C) for 30 min to extract DNA
from the double membrane-enclosed EVs. The DNA within the subsequent
supernatant was extracted according to the instructions provided in the
DNeasy PowerSoil kit (QIAGEN, Germany) and quantified using the QIAxpert
(QIAGEN, Germany) software. The V3-V4 hypervariable regions of the 16S
rDNA contained in each sample were amplified and the libraries were
prepared and quantified as described previously13. All
resulting 16S rDNA amplicons were sequenced using the MiSeq (Illumina,
USA).