Fecal Microbiome Profiling in Mice

Mouse feces were collected by placing mice in metabolic cages (Hatteras Instruments, Cary, NC, USA) for a few hours. Then, feces (0.25 g) from each mouse in different cages were collected for microbiome analysis to avoid the influence of allocoprophagy (a habit of mice that ingest feces from other mice) on fecal microbiota analysis following previous publications [93 (link),98 (link),99 (link)]. Briefly, the metagenomic DNA was extracted from the prepared samples using a DNAeasy Kit (Qiagen, Redwood City, CA, USA). The quality and concentration of the extracted DNA were measured by nanodrop spectrophotometry. Universal prokaryotic primers 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) with appended 50 Illumina adapter and 30 Golay barcode sequences were used for 16S rRNA gene V4 library construction in Miseq300 platform (Illumina, San Diego, CA, USA). The raw sequences and operational taxonomic unit (OTU) were classified following Mothur’s Standard Operating Procedures (SOP) [100 (link)].