Illumina-Seq of mouse: adult male stool
Caecal contents were collected from individual mice following 12 weeks intervention. Total metagenomic DNA was extracted from caecal contents with the QIamp® PowerFecal® DNA Kit (Qiagen, Milano, Italy) where, an additional bead beating step was incorporated into the protocol. Extracted DNA was quantified using the NanoDropTM 8000 Spectrophotometer (Thermo Fisher Scientific). Total genomic DNA was then subjected to PCR amplification by targeting a 464-bp fragment of the 16S rRNA variable region V3-V4 (Baker et al., 2003, Claesson et al., 2010) using the specific bacterial primer set 341F (5'- CCTACGGGNGGCWGCAG -3') and 806R (5'- GACTACNVGGGTWTCTAATCC -3') with overhang Illumina adapters. Unique barcodes were attached to the forward primer for facilitating the differentiation of samples. Amplicons were cleaned with the Agencourt AMPure kit (Beckman coulter) following the manufacturer's instructions, and DNA was quantified using the Quant-iT PicoGreen dsDNA kit (Invitrogen). Amplicons were mixed and combined in equimolar ratios, and the quality and purity of the library was checked with the High Sensitivity DNA Kit (Agilent) by the Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA). The library was sequenced on an Illumina MiSeq platform at CIBIO (Center of Integrative Biology) – University of Trento (Trento, Italy).