16S of gut goat metagenome. Feces sample, goat: 4334 diet: Q4, sampleID: 28
Bacterial/Archeal DNA was amplified using the primers described in literature (Caporaso et al., 2011) which target the V3V4 hypervariable regions of the 16S rRNA gene. The PCR amplifications were performed in 25 L volume per sample. A total of 12.5 L of KAPA HIFI Master Mix 2 (Kapa Biosystems, Inc., Wilmington, MA, USA) and 0.2 L of each primer (100 M) were added to 2 L of genomic DNA (5 ng/L). Blank controls (i.e., no DNA template added to the reaction) were also performed. A first amplification step was performed in an Applied Biosystem 2,700 thermal cycler (ThermoFisher Scientific, Waltham, MA, USA); the samples were denatured at 95 C for 5 min, followed by 25 cycles with a denaturing step at 95 C for 1 min, annealing at 56 C for 1 min and extension at 72 C for 1 min, with a final extension at 72 C for 7 min. Amplicons were cleaned with Agencourt AMPure XP (Beckman, Coulter Brea, CA, USA) and libraries were prepared following  the Metagenomic Sequencing Library Preparation Protocol (Illumina, San Diego, CA, USA). The libraries obtained were quantified by Real Time PCR with KAPA Library Quantification Kits (Kapa Biosystems, Inc., MA, USA), pooled in equimolar proportion and sequenced in MiSeq (Illumina) run with 2  250-base paired-end reads each.