Gut Microbiome Profiling by 16S rRNA Sequencing

Unflushed cecal tissue and content was suspended in 1.4 ml ASL buffer (Qiagen) and homogenized with 2.8 mm ceramic beads followed by 0.5 mm glass beads using an OMNI Bead Ruptor (OMNI International). DNA was extracted from the entire resulting suspension using QIAamp DNA Stool Mini Kit (Qiagen) according to manufacturer’s protocol. DNA was quantified using Qubit broad range DNA assay (Life Technologies). The V4 region of 16s rRNA gene was amplified using universal primers (515f and 806r) (Caporaso et al., 2012 (link)). Individual samples were barcoded, pooled to construct the sequencing library, and then sequenced using an Illumina Miseq (Illumina, San Diego, CA, United States) to generate pair-ended 250 nt reads. Quantitative PCR was performed for A. muciniphila as described in Schneeberger et al. (2015) (link) with DNA for standard curve isolated from the cultivated microbe.