Quantification of SFB and Bacteroides

To quantify relative levels of specific bacteria by qPCR, genomic DNA was extracted from mucosal scrapings, flushed lumenal contents, or feces using the QIAamp Fast DNA Stool Mini Kit. To obtain ileum lumen samples, the distal 10 cm of the ileum was flushed with 3 ml sterile PBS into an eppendorf, centrifuged at 20,000 g for 15 mins, 4°C. Supernatant was discarded and pellets were res uspended in 1.0 ml InhibitEX Buffer (Qiagen) and frozen at −20°C until use. To o btain ileum mucosal scrapings, flushed ileum segments were opened longitudinally on a sterile petri dish, then mucosa and associated bacteria were scraped and frozen in 1.0 ml InhibitEX Buffer. To separate PP and villi-associated mucosa, 0.5 cm segments of PPs and surrounding tissues were excised from flushed ileum tissues, opened longitudinally on a sterile petri dish, and the mucosa from the tops of PP domes were scraped and frozen in 1.0 ml of InhibitEX Buffer. The remaining villi segments of flushed ileums were opened longitudinally, and villi-associated mucosa were scraped and frozen in 1.0 ml of InhibitEX Buffer. For feces, 1–2 freshly collected fecal pellets were frozen at −20°C until use. Upon thawing, mucosal scrapings, l uminal contents, and feces were homogenized in a 2 ml eppendorf containing 400 ul of 0.5 mm glass acid-washed beads (Sigma) and up to 1.4 ml of InhibitEX buffer. Samples were heated at 90°C for 1 hour with vigorous vortexing, then centrifuged at 20,000 g for 3 mins. After spinning, 600 ul of supernatant was combined with 600 ul Buffer AL (Qiagen) and 25 ul Proteinase K (Qiagen), then heated for 70°C for 20 min. After heating, 600 ul ethanol was added to each sample, then samples were applied to columns, washed with 600 ul Buffer AW1 and 600 ul Buffer AW2 (Qiagen) in sequential steps, and eluted in 100 ul Buffer ATE (Qiagen). qPCR was conducted on samples to determine the relative amount of SFB, Bacteroides fragilis, and total Eubacteria using SYBR Green reagent and bacteria-specific primers on a LightCycler-96 RT-PCR System (Weber Scientific). Expression levels were normalized to 16S using the ΔCt method.