Fecal Microbiome Analysis in Mice

Feces from nine mice (0.25 g per mouse) from different cages in each experimental group were divided into three samples per group (three mice per sample) before performing microbiota analysis following a previous protocol [41 (link)]. In short, metagenomic DNA was extracted from 0.25 g feces by DNeasy PowerSoil Kit (Qiagen, Germantown, MD, USA). The Universal prokaryotic 515F (forward; (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (reverse; 5′-GGACTACHVGGGTWTCTAAT-3′), with appended Illumina adapter and Golay barcode sequences, were used for 16S rRNA gene V4 library construction and sequenced using Miseq300 platform (Illumina, San Diego, CA, USA) at Omics Sciences and Bioinformatics Center, and Microbiome Research Unit for Probiotics in Food and Cosmetics, Chulalongkorn University. Raw sequences were quality processed and operational taxonomic unit (OTU) classified following Mothur’s standard operating platform procedures [42 (link),43 (link)]. Bioinformatic analyses included good’s coverage, alpha diversity (e.g., Chao), and beta diversity (e.g., non-metric multidimensional scaling (NMDS)). Linear discriminant analysis effect size (LEfSe) and meta-stats were also performed to determine species marker and unique representing species of the interested group, respectively [42 (link),44 (link)].