title: Bovine CTR group sample n.153_S153
design: A total of 12.5 L of Phusion High-Fidelity Master Mix 2x (ThermoFisher Scientific, Waltham, MA, USA) and 0.2 L of each primer (100 M) were added to 2 L of genomic DNA (5 ng/L). A first amplification step was performed in an Applied Biosystem 2,700 thermal cycler (ThermoFisher Scientific). Samples were denatured at 98 C for 3 min, followed by 25 cycles with a denaturing step at 98 C for 30 s, 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 16S Metagenomic Sequencing Library Preparation Protocol (Illumina, San Diego, CA, USA). The libraries obtained were quantified by RealTime PCR with KAPA Library Quantification Kits (Kapa Biosystems, Inc., MA, USA), pooled in equimolar proportion, and sequenced in three different MiSeq (Illumina) runs with 2  402-base paired-end reads each.