Gut Microbiome Profiling in Mice

Mice were divided into two groups. Mice in each group were fed a CD or an ID for 14 days before feces collection. On day 15, all fecal samples obtained from mice of both groups were collected and stored at -80 °C until analyses of the gut microbial flora. A two-step tailed PCR method was used for the preparation of dsDNA libraries. Library concentrations were measured with a Synergy H1 microplate reader (BioTek) and a QuantiFluor dsDNA System (Promega). The library quality was assessed using a Fragment Analyzer (Advanced Analytical Technologies, Ankeny, IA, USA) with a dsDNA 915 Reagent Kit (Agilent, Santa Clara, CA, USA). Paired-end sequencing (2 × 300 bp) was performed on the Illumina MiSeq platform (Illumina, San Diego, CA, USA) with the MiSeq Reagent Kit v3 (Illumina). A sequence that completely matched the primer used was extracted by using the fast barcode splitter tool. After the trimming of the primer sequence, denoized sequences were analyzed using Qiime2.0 (2019.4). The EzBioCloud 16S database (28 (link)) was used to classify the bacterial species. These analysis procedures were performed at Bioengineering Lab. Co., Ltd. (Sagamihara, Kanagawa, Japan).