title: 16s RNA-seq of mus musculus: adult female feces
design: DNA extraction was performed on 136 feces samples to obtain information on intestinal microbiota changes using the PowerSoil DNA Isolation Kit (MO BIO, Cat.No.12888). To amplify 16S rRNA genes from distinct regions, specific primers with barcodes were used, and PCR reactions were performed using Phusion High-Fidelity PCR Master Mix (New England Biolabs). TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, USA) was used to generate the sequencing libraries, which were subsequently sequenced on an Ion S5TM platform to yield 400 bp/600 bp single-end reads. After analyzing sequences using the Uparse software (Uparse V8.1.1861), OTUs (Operational Taxonomic Units) were obtained. To obtain species information, the SILVA database was used as a reference to annotate representative sequences in OTUs. Multiple sequence alignments were performed using the MUSCLE software (Version 3.8.31) to determine the phylogenetic relationship of different OTUs and the differences in the dominant species in different samples (groups).