2.5. IgA sequencing (IgA-seq)

Mouse feces were homogenized on a 70-μm cell strainer (Greiner Bio-One) using 1% BSA in PBS. After brief centrifugation at 500 × g for 5 min at 4°C to remove large debris, the supernatant was centrifuged at 12,000 × g for 5 min to pellet the bacteria. The bacterial pellet was resuspended in 1% BSA in PBS containing biotinylated goat anti-mouse IgA (BD Biosciences). After washing with 1% BSA in PBS, the bacteria were stained with streptavidin-BV421 (BioLegend). The bacteria were washed and resuspended in 1% BSA/PBS containing SYTORed (Thermo Fisher Scientific), prior to flow cytometry. Flow cytometric analysis was performed using a FACSAria III flow cytometer with DIVA v9.0 (BD Biosciences), and data were analyzed using FlowJo version 10.9 (BD Biosciences). IgA-binding bacteria were magnetically enriched using a MojoSort system (BioLegend) with streptavidin nanobeads (BioLegend), according to the manufacturer’s instructions.