16S rRNA of mouse: stool
Fecal pellets were collected from 812-week-old mice and stored at 80C prior to DNA extraction. DNA was isolated using the DNeasy PowerSoil Pro Kit with bead-beating on a PowerLyzer 24 Homogenizer, following the manufacturer's instructions with minor modifications. Extracted DNA was stored at 20C until sequencing. 16S rRNA gene amplicon sequencing of the V3V4 region (primers 341F785R) was performed by LGC Genomics using Illumina MiSeq v3 chemistry. PCR was conducted with barcoded primers based on Klindworth et al. with a modified 785R primer. Amplicons were pooled, purified with AMPure XP and MinElute columns, and libraries were constructed using the Ovation Rapid DR Multiplex System. Libraries were pooled and size-selected before sequencing. Sequencing quality was verified using the ZymoBIOMICS Microbial Community DNA Standard.