Rumen and Fecal Microbiome DNA Extraction and 16S Sequencing

Genomic DNA from rumen and fecal samples was extracted using the Mag-Bind Stool DNA Kit (Omega Bio-Tek, Norcross, GA, U.S.), according to the manufacturer’s protocol with a minor modification. For the lysing step, the protocol was adapted to use a mixer mill (Retsch MM 200) set up for 10 min at 25 Hz. The quality of the resulting DNA was checked using gel electrophoresis (1% agarose gel) and the concentration was measured with a NanoDrop One spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Bacterial amplicon libraries of the V4 region from the 16S rRNA gene were prepared as described by Paz et al. [20 (link)]. The resulting amplicons were normalized to a concentration of 1–2 ng/μl using the SequalPrep^TM normalization plate. Pooled libraries were sequenced using the Illumina MiSeq platform (Illumina, San Diego, CA, USA). Raw sequences are available at the NCBI Sequence Read Archive (SRA) under the accession no. SRP271418.