16S sequencing from content of cecum sample from SPF mouse
We sampled five SPF mice, both mucus and luminal content separately in five sites: duodenum, jejunum, ileum, cecum and colon. Following DNA extraction (QIAamp PowerFecal DNA Kit, Qiagen), amplicons spanning the 16S ribosomal RNA (rRNA) gene variable region 3 and 4 (V3-V4) were prepared using degenerate primers 515FY and 806R according to the protocols benchmarked for the Earth Microbiome project. The PCR amplified amplicons were gel purified (MinElute Gel Extraction Kit, Qiagen), and Illumina sequencing adaptors and barcodes were added followed by bead-based clean up (AMPure beads, Beckman Coulter).