3044BPX32
DNA was extracted from composite fecal samples collected during each cows stay in the head chambers, equating to 4 samples per animal over the course of the study. Total DNA was extracted from 0.25 mg of homogenized fecal samples using DNeasy PowerLyzer PowerSoil Kit (QIAGEN, Hilden, Germany). In the purification process, samples were homogenized, cells lysed, inhibitors removed, and DNA bound and eluted following manufacturer guidelines. Extracted DNA and quality of the DNA extracted was analyzed with Qubit 3.0 (Thermo Fisher Scientific, Waltham, MA, USA). Bacterial abundances were determined by amplifying the V4 region of the 16S rRNA gene using forward 515F and reverse barcoded 806R primers (Apprill et al., 2015; Parada et al., 2016). Extracted DNA was stored at -20 C prior to PCR amplification which was carried out using Phusion Hot Start II High-Fidelity PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). PCR was run in triplicate and triplicates of each sample were combined and quantified by Qubit 3.0. Each sample was then pooled with equimolar concentrations (100 ng DNA / sample) into a verified index library in a single sterile tube and purified using the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany). DNA was visualized on a 1.5% agarose gel for quality assurance and nuclease free water was also amplified with samples and sequenced as a control for kit contamination. The final library was sequenced at the DNA Technologies Core Facility of the Genome Center at University of California, Davis on the Illumina Miseq 500 PE250 platform.