Normal rat serum

Samples were processed as described previously (17 (link)). We centrifuged glycerol-stored stool samples at 50 × g at 4°C for 15 min and then washed them three times in 1 ml PBS/1% BSA at 8,000 × g for 5 min. We collected the presort fraction as 20 μl after resuspension before the final wash and stored the washed samples at −80°C. We then resuspended the cell pellet in 25 μl of 20% normal rat serum (Jackson ImmunoResearch) in PBS/1% bovine serum albumin (BSA) and incubated the samples for 20 min on ice. After incubation, we added 25 μl of 1:12.5 α-mouse-IgA-PE (eBioscience; clone mA-6E1) to each sample and incubated samples on ice for 30 min. Finally, we washed samples three times in 1 ml PBS/1% BSA, resuspended them in PBS/1% BSA, and transferred them to blue filter-cap tubes (VWR 21008-948) for flow sorting. We sorted an average of 50,000 cells from the IgA-positive and IgA-negative bacteria into sterile microcentrifuge tubes on the BD FACSAria II at the MIT Koch Institute Flow Cytometry Core (Cambridge, MA). We then centrifuged the samples, removed the supernatants, and resuspended the pellets in a final volume of 10 μl of sheath fluid. Samples were stored at −80°C until DNA library prep, in which 2 μl (∼10,000 cells) was used directly as the template for PCR.