16S rRNA gene amplicon sequencing of dairy cattle rumen content and epithelial microbial communities
genomic DNA from each sample was amplified using Platinum Taq DNA Polymerase (Thermo Fisher Scientific, Waltham, MA) with one replicate per sample using universal 16S rRNA gene bacterial primers  [515F (5-GTGYCAGCMGCCGCGGTAA-3; Parada et al., 2016 ), and 806R (5-GGACTACNVGGGTWTCTAAT-3; Apprill et al., 2015 )] for the variable region V4, as previously described (Kozich et al., 2013). All samples underwent PCR with an initial denaturation step at 94C for 3 min, followed by 45 s of denaturing at 94C, 20 s of annealing at 50C, and 90 s of extension at 72C. This was repeated for 35 total PCR cycles and finished with a 10 min extension at 72C. All the PCR products were then purified with the QIAquick 96 PCR Purification Kit (Qiagen Sciences Inc, Germantown, MD) according to the manufacturers recommendations. PCR bar-coded amplicons were mixed at equal molar ratios and used for Illumina MiSeq paired-end sequencing with 150 bp read length and cluster generation with 10% PhiX control DNA on an Illumina MiSeq platform (Illumina Inc., San Diego, CA).