DNA extraction, PCR, and sequencing

DNA was extracted from ruminal fluid post-fermentation. The procedure of the DNA extraction method was similar to that previously described by Yu and Morrison [34]. After the chemical/mechanical cell lysis and isopropanol precipitation of nucleic acids, metagenomic DNA was purified with Rnase and proteinase K treatment, followed by the use of QIAamp columns from the Qiagen DNA Stool Mini Kit (Qiagen, Hilden, Germany). Genomic DNA concentration was determined using a Nanodrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE), and verified using Invitrogen Qubit fluorometer with PicoGreen (ThermoFisher Scientific, Wilmington, DE). Extractions were stored at -20˚C until sequencing library preparation. Bacterial 16S rRNA genes were PCR-amplified with dual-barcoded primers targeting the V4 region, as per the protocol of Kozich [35]. Amplicons were sequenced with an Illumina MiSeq using the 250-bp paired-end kit (v.2). Sequences were denoised, taxonomically classified using Greengenes (v. 13_8) as the reference database, and clustered into 97% similarity operational taxonomic units (OTUs) with the mothur software package (v. 1.39.5), as previously described by Schloss, Westcott [36], and following the manufacturer-recommended procedure (https://www.mothur.org/wiki/MiSeq_SOP; accessed November 2017). Sequence files are available from the NCBI Sequence Read Archive (SRA Accession SRP150716). Additional descriptive information is associated with NCBI BioProject PRJNA476124.