SPFvsGF_5R16S rRNA

Specified Pathogen Free (SPF, n=16) and germ free (GF, n=16) mice (C57BL/6) were purchased from Kunshan Mingqian Microbiology Research Institute（Kunshan, China）。 To prevent contamination of GF mice during transportation and maintain consistent sampling environments for both groups of samples, ovarian sampling was performed in a sterile manner in the laboratory's ultra clean workbench of the purchasing company. After ovarian collection, it was sealed and transported in dry ice to Hangzhou Lianchuan Biotechnology Co., Ltd.

(Hangzhou, China) for 5R 16S rRNA gene sequencing. The DNA of ovarian tissue was extracted using a bacterial genome extraction kit (TIANGEN, DP302-02). Design primers targeting the variable regions of 16S rDNA V2, V3, V5, V6, and V8 (5R primers; Supplementary Table 1) (5R primers: V2 forward TGGGAACGGGTGTAAA, V2 reverse CCGTGTCTCAGTCCCARTG; V3 forward - ACTCCTACGGGAGGCAGC, V3 reverse - GTATTACCGCGGCTGCTG; V5 forward - GTGTAGGGTGRAATGCG, V5 reverse - CCCGTCAATTCMTTGAGTT; V6 forward - GGAGCATGTGGWTTAATTCGA, V6 reverse - CGTTGCGGGACTTAACCC; V8 Forward - GGAGGAAGGTGGGGATGAC, V8 Reverse - AAGGCCCGGGAACGTATT; ）Perform PCR amplification, purify and quantify the PCR products using AMPure XP beads (Beckman Coulter Genomics, Danvers, MA, USA), and perform high-throughput sequencing (NovaSeq 6000 SP Reagent Kit; Illumina NovaSeq 6000).