title: DNA-Seq of fermentation fluid: dairy cow ruminal fluid
design: Bacterial DNA was extracted from 48 h samples using an Omega Stool DNA kit (Omega Bio-Tek, Norcross, GA, USA). The quality and quantity of the DNA were determined using NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wil-mington, DE, USA). Amplicon library preparation was performed by polymerase chain reaction (PCR) of the V3-V4 region of the 16s rRNA gene using universal primers 338F (5-ACTCCTACGGGAGGCAGCAG-3), and reverse primers 306R (5-GGACTACHVGGGTWTCTAAT-3) (Bi et al. 2018). The 20 L reaction system included 10 ng of template DNA, 4 L of FasPfu buffer, 2 L of 2.5 mmol/L dNTPs, 0.8 L of each primer, 0.4 L of FasPfu polymerase, 0.2 L bovine serum albumin, and doudle dis-tilled H2O to 20 L. The amplicons were electrophoresed on 4% agarose gel, purified using an Agencourt AM Pure XP kit (Beckman Coulter Genomics, Indianapolis, IN, USA), and quantified using the QuantiFluorTM-ST system (Promega, Madison, WI, USA). Finally, the purified amplicons were pooled in equimolar concentrations and pair-end sequenced on an Illumina MiSeq platform (Illumina, Inc., San Diego, CA, USA).