title: Rumen goat microbiota (Archaea) Sample: 4, Diet: Standard, Time point: T0
design: Effects of different diets on microbiome diversity of rumen liquor in dairy goats was evaluated on a total of 18 Alpine goats, divided in three diet groups (Standard diet, Hemp supplement, Linseed supplement). Two samples per goat were taken, one at T0 and one at T1 (after about 140 days). For metagenomic analysis, the archaeal 16S rRNA gene was amplified using primers described in literature (Kittelman et al., 2013), which target the V3-V4 hypervariable regions. All PCR amplifications were performed in 25 μl, using 0.2 µl of each primer (100 µM) and 2 µl of genomic DNA (5 ng/µl). PCR amplification was performed as follows: initial denaturation at 98 °C (30 seconds); then, 25 cycles composed of a denaturing step (98 °C for 30 seconds), annealing (56 °C for 1 minute) and extension (72 °C for 1 minute); final extension at 72 °C for 7 minutes. Amplicons were cleaned-up following Illumina 16S Metagenomic Sequencing Library Preparation protocol (http://supportres.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf). Libraries were prepared by a second PCR amplification step using Nextera XT index 1 Primers (N7XX) and Nextera XT Index 2 Primers (S5XX) from the Nextera XT Index Kit (FC-131-1001 or FC-131-1002), following 16S Metagenomic Sequencing Library Preparation protocol (http://supportres.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf). The libraries obtained were quantified and pooled in equimolar proportion and sequenced in one Miseq (Illumina) run with 2 x 300-base paired-end reads.