16S rRNA gene sequencing. Bovine uterine metagenome
A manual precipitation protocol was used for DNA extraction. Library construction and sequencing were performed by the University of Missouri DNA Core. A Qubit dsDNA BR Assay (Life Technologies, Carlsbad, CA) was used to determine DNA concentration. Samples were normalized to 3.51 ng/L DNA for PCR amplification. The V4 hypervariable region of the 16S rRNA gene was amplified using single-indexed universal primers [U515F (GTGCCAGCMGCCGCGGTAA); 806R (GGACTACHVGGGTWTCTAAT)] with standard adapter sequences (Illumina Inc., San Diego, CA). The PCR program for amplification was: 98 C (3:00) + [98 C (0:15) + 50 C (0:30) + 72 C (0:30)]  40 cycles + 72 C (7:00; min:s). The V4 region of the 16S rRNA gene was selected for library generation because this region yields optimal community clustering. The Illumina MiSeq platform (V2 chemistry with 2  250-bp paired-end reads) was used to sequence pooled amplicons.