Gut Microbiome Analysis of Mice

The 5 mice were selected from Ex and MCP-M groups at random, and DNA from feces was extracted by using a Genomic DNA Kit (Omega Bio-tek, Inc., Norcross, GA, USA). The 16S rRNA genes (V3-V4 regions) were amplified from the whole genome via the primer pair (341 F, 5′- CCTAYGGGRBGCASCAG-3′; 806R, 5′-GGACTACHVGGGTWTCTAAT-3′). All the amplicons were purified, quantified, and sequenced on an Illumina novaseq platform (San Diego, CA, USA). The barcode and connector sequence were removed. FLASH (v1.2.8) was used to stitch the double-ended sequences, and Vsearch (v2.3.4) was used to filter out the unqualified sequences (8 (link)). Finally, the sequence with 97% similarity was classified as an OTU. 16S rRNA gene reads were down-sampled to a read depth of 44,367 reads/sample and reads mapped to 16S OTUs (1,635 reads), to ensure sample compatibility regardless of sampling depth (21 (link)). Gene functions were predicted by PICRUSt.2 and analyzed by KEGG pathway enrichment analysis.¹