NGS of fecal sample of Fischer 344 rat: 2yr-F-C1
V3-V4 region of 16S rRNA was amplified with PCR. The primers were 341F and 805R. PCR products were confirmed with electrophoresis and purified using QIAquick PCR purification kit (Qiagen, Valencia, CA, USA). The purified PCR products were tagged with Illumina indices and adapters from Nextera XT Index Kit (Illumina, San Diego, CA, USA). Short DNA fragments were eliminated using FavorPrep DNA purification kit (Favorgen, Taiwan). The PCR amplicons were quantified by Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific, Wilmington, DE, USA). After pooling of 300 ng per sample, the PCR products were purified with a FavorPrep DNA gel extraction kit. The quality assessment for confirmation of DNA integrity and product size was conducted on a Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA) using a DNA 7500 chip at ChunLab, Inc. (Seoul, South Korea).