Gut Microbiome Analysis of Mice

Bacterial DNA was isolated from feces using the PowerFecal Pro DNA Kit (QIAGEN). DNA from two mice was combined in equivalent proportions (n = 3–4/group) for 16S rRNA sequencing. The samples were purified and sequenced using the iSeq platform (Illumina, San Diego, CA, USA). Trimmomatic (ver. 0.39) was used to clean the sequences, and then the data were organized into operational taxonomic units and aligned with QIIME2 (version 1.9.5) (accessed on 15 December 2022) [30 (link)]. In addition, α- and β-diversity analyses were conducted using QIIME2. Quantitative PCR was used to validate the results of 16S rRNA sequencing and to quantify the abundance of 10 well-known probiotics in fecal bacteria (CFX System, Bio-Rad). Bacterial DNA was amplified using the specific bacterial primers listed in Supplemental Table S2 (Macrogen). The ratio of total bacteria was used to calculate the relative abundance (F341/R518) (n = 10 per group) [31 (link)].