16S rRNA seq of mouse: stool day 0
Mouse fecal samples were obtained by gently holding each mouse over a clean Petri dish within a laminar flow hood to encourage natural defecation. The samples were promptly placed in sterile tubes, quickly frozen on dry ice, and stored at 80 C for up to 2 wk before analysis. Bacterial sequencing analysis was carried out at the University of Michigan Host Microbiome Core facility. The V4 region of the 16S rRNA gene was amplified from each sample utilizing a dual-indexing sequencing strategy. PCR amplification conditions were as follows: initial denaturation at 95 C for 2 min, followed by 30 cycles of 95 C for 20 s, 55 C for 15 s, and a final extension at 72 C for 10 min. Accuprime High Fidelity Taq (ThermoFisher, Grand Island, NY) was used for amplification. The PCR-amplified samples were frozen at 20 C until further use. Amplicons were visualized on an eGel 96 SYBR Safe 2% gel system (ThermoFisher) and normalized with the SequalPrep Normalization Plate Kit (ThermoFisher). Sequencing was performed on the Illumina MiSeq platform with the MiSeq Reagent Kit V2 (500 cycles), following the manufacturers protocol.