*2.7. Microbiota Analysis*

The fecal DNA of mice was extracted using a commercial E.Z.N.A.® DNA kit from Omega Bio-Tek Inc. (Norcross, GA, USA). DNA concentration and purity were determined via NanoDrop2000 from Thermo Fisher Scientific Inc. (Pittsburgh, PA, USA). The 1% agarose gel electrophoresis was used for DNA quality assay. A thermocycler PCR system (ABI GeneAmp® 9700, Thermo Fisher Scientific, Waltham, MA, USA) was adopted for the amplification of 16 S rRNA gene in the hypervariable region of bacterial V3-V4, conducting with the universal primers 338F (5 -ACTCCTACGGGA GGCAGCAG-3 ) and 806R (5 -GGACTACHVGGGTWTCTAAT-3 ). An AxyPrep DNA gel extraction kit (Axygen Inc., Corning, NY, USA) was applied to purify the amplified products, and a QuantusTM fluorometer (Promega Inc., Madison, WI, USA) was used for the quantification of the purified products. An Illumina MiSeq System (Illumina Inc., San Diego, CA, USA) was adopted

for the paired-end sequencing of amplicons. The raw Illumina data was quality-filtered and merged using FASTQ version 0.20.0 software and FLASH version 1.2.7 software, respectively. Sequences with more than 97% similarity were clustered into the same amplicon sequence variants (ASVs) by DADA2 plugin of Qiime2 version 2020.2 software. The ASVs taxonomic assignments were performed using the naïve Bayes consensus taxonomy classifier based on SILVA 16S rRNA database (v 138). The analysis of 16S rRNA microbiome sequencing data was conducted by bioinformatic tools on Majorbio i-Sanger cloud platform (http://en.majorbio.com). The alpha-diversity and beta-diversity analysis, Kruskal–Wallis H test, and linear discriminant analysis effect size (LEfSe) analysis were performed based on the Majorbio i-Sanger cloud platform.
