Rumen Microbiome DNA Extraction and 16S Sequencing

One milliliter of rumen sample was centrifuged at 14,000× g. The remaining pellets were used for DNA extraction by i-genomic Stool DNA Extraction Mini Kit (iNtRON Biotechnology, Inc., Korea) according to the manufacturer’s instructions. DNA was eluted in 50 μL elution buffer, and DNA quantity and quality were checked by agarose gel electrophoresis and nanodrop spectrophotometer (Thermo Fisher Scientific, Madison, Wisconsin, USA). The V4 region of the bacterial 16S ribosomal DNA gene was amplified using primers 515F and 926R [33 (link)]. PCR amplification was conducted under the following conditions: 94°C for 3 min; 35 cycles of 94°C for 45 s, 50°C for 60 s, and 72°C for 90 s; and 72°C for 10 min. PCR products purification and preparation for sequencing using Illumina MiSeq system were conducted according to the protocol described by Comeau et al. [34 ] in Integrated Microbiome Resource (Dalhousie University, Canada).