Rat intestine microbiota, Sample I5
Total bacterial DNA extraction was performed using the QIAamp DNA Microbiome Kit (QIAGEN, Hilden, Germany), following manufacturer’s instructions. Particular attention was paid to avoid environmental contamination of collected samples, and cross-contamination between samples. 16S rRNA gene amplicon libraries targeting V3-V4 hypervariable regions were performed with a two-step barcoding approach according to Illumina 16S Metagenomic Sequencing Library Preparation (Illumina, San Diego, CA, USA). Library concentration and exact product size were measured using a KAPA Library Quantification Kit (Kapa Biosystems, Woburn, MA, USA) and an Agilent 2100 Bioanalyzer System (Agilent, Santa Clara, CA, USA), respectively. Prior to sequence, libraries were pooled using Amicon Ultra 0.5ml Centrifugal Filters (Merck Millipore Ltd, Cork, Ireland). The resulting library was loaded on a MiSeq 500 cycle-v2 cartridge to obtain a paired-end 2×250 bp sequencing.