Platinum pfx

Methanol Carnoy’s fixed intestinal segments were paraffin embedded prior to microscope slide generation. Slides were deparaffinized in xylene, twice incubated in 100% ethanol, and retreated with xylene prior to drying briefly and placing in a dessicator for approximately 1 hour. Mucus layer (~30 μm immediately adjacent to epithelium) or lumen-associated (middle of lumen) samples were taken for each intestinal segment (ileum, cecum, and distal colon) of the Rag1^−/− mice using an Arcturus Veritas microdissection instrument (Applied Biosystems). DNA was extracted from each sample with the PicoPure DNA extraction kit (Applied Biosystems). Prior to qPCR for quantifying relative abundance of each strain, a PCR for non-specific amplification of the barcoding vector was employed (primers are listed in Table S1). 10 ng of extracted DNA were amplified in duplicate for each sample with Platinum Pfx (Invitrogen) with the following protocol: 95 °C for 5 minutes; 20 cycles of: 95 °C for 30 seconds, 58 °C for 30 seconds, and 72 °C for 30 seconds; final extension at 72 °C for 10 minutes. After PCR, duplicate samples were pooled and purified using Qiagen MinElute PCR Purification Kit and diluted (ileum diluted 1:10, colon segments diluted 1:100) prior to use in qPCR.