title: 16S rRNA gene amplicon sequencing (V3-V4 region)
design: Bacterial DNA was extracted from 100 mg small intestinal content per sample using the QIAamp PowerFecal DNA Kit (Qiagen, Hilden, Germany) and further purified using AMPure XP beads (Beckman Coulter, Brea, CA, USA) according to the manufacturers protocols. The concentration of genomic DNA was measured using a Qubit 2.0 Fluorometer with Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Bacterial DNA was amplified with tagged primers (5-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG and 5-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C), covering the V3-V4 region of the bacterial 16S rRNA gene. PCR and DNA purifications were performed according to Illuminas demonstrated protocol (Part # 15044223 Rev. B). The PCR product libraries were quantified and qualified by using DNA 1000 Kit on Agilent 2100 Bioanalyzer instrument (Agilent Technologies, Waldbronn, Germany). Equimolar concentrations of libraries were pooled and sequenced on an Illumina MiSeq platform (Illumina, San Diego, CA, USA) using MiSeq Reagent Kit v3 (600 cycles PE)