Gut Microbiome Profiling in Mice

Colon luminal contents in mice were collected immediately after euthanasia and were rapidly flash-frozen in liquid nitrogen for 30 min before storing at −80°C. Total bacterial DNA was extracted by using a Magnetic soil and stool DNA kit (TIANGEN, China). The V3-V4 region of 16S rRNA gene was amplified using PCR assay with. The amplicon primer sequences were as follows: forward primer 5′-(GTGCCAGCMGCCGCGGTAA)-3′; reverse primer 5′-(GGACTACHVGGGTWTCTAAT)-3′. The libraries were constructed using NEB next ultra-library prep kit (Illumina, USA) and paired-end sequenced on Illumina Novaseq6000 platform (Illumina, USA) of Novogene Co., Ltd. (Beijing, China). The filtering of raw sequence and taxonomic classification were performed as described previously (Li et al., 2020 (link)). The diagrams of alpha and beta diversity and relative abundance of microbiota were drawn using the R packages ggplot2 and vegan. The significance of beta diversity based on Weighted UniFrac dissimilarity was tested by analysis of similarities (Anosim) within the R package vegan.