Metagenomics of mouse
aDNA from feces kept at -80 oC were extracted by Qiagen DNeasy PowerSoil kits following the standard protocol. The V3-V4 domains of the 16S rRNA were amplified using primers 319F/806R ([TCGTCGGCAGCGTCAGATGTGT-ATAAGAGACAG(spacer)GTACTCCTACGGGAGGCAGCAGT] and [GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG(spacer)-CCGGACTACNVGGGTWTCTAAT] respectively) containing an Illumina tag sequence, a variable length spacer, linker sequence, and the 16S target sequence. Each sample was barcoded with an Illumina P5 adapter sequence, a unique 8 nucleotide (nt) barcode, a partial matching sequence of the forward primer, and reverse primers with an Illumina P7 adapter sequence, unique 8 nt barcode, and a partial matching sequence of the reverse adapter. The final product was quantified on a Qubit 4.0 instrument using the dsDNA Broad Range DNA kit (Invitrogen, Carlsbad, CA) and individual amplicons were pooled in equal amounts. The pooled library was cleaned with Ampure XP beads (Beckman Coulter, Brea, CA) and bands of interest were further isolated by gel electrophoresis (Sage Science, Beverly, MA). The library was quantified via qPCR then sequenced with 300 base pair, dual end sequencing with an Illumina MiSeq