Fecal microbiota 16S rRNA sequencing of aged donors and FMT recipients in a neonatal overfeeding mouse model

We first employed an established neonatal overfeeding model by reducing litter size, assigning litters to either 4 pups per dam (Overfed group) or 8 pups per dam (Normal-fed control group). For the FMT experiment, donor fecal material was collected from 24-month-old normal-fed or overfed mice and transplanted into 12-month-old recipients maintained strictly on a standard chow diet (rNormal-Chow and rOver-Chow), fecal samples were prospectively collected at the end of the intervention.

Microbial genomic DNA was extracted from fecal samples using the DNA Stool Mini Kit (TIANGEN). The V3-V4 hypervariable regions of the 16S rRNA gene were amplified using universal primers (515F/806R). Sequencing libraries were constructed with the NEB Next® Ultra™ II FS DNA PCR-free Library Prep Kit (New England Biolabs, E7430L) to ensure high-quality, bias-free amplicons.