title: DNA-Seq of fermentation fluid: dairy cow ruminal fluid
design: Total DNA of WPCS was extracted by the Phusion High-Fidelity PCR Master Mix with GC Buffer kit (New England Biolabs) according to the manufacturers protocol. The DNA extract was analyzed on a 1% agarose gel, and the concentration was quantified using a NanoDrop 2000 UVvis spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). The hypervariable region V3-V4 of 16S rRNA gene was amplified with primer pairs 515F (GTGCCAGCMGCCGCGGTAA) and 806R (GGACTACHVGGGTWTCTAAT) [13] using an ABI GeneAmp 9700 PCR thermocycler (ABI, Foster City, CA, USA), and the primers ITS51737F (GGAAGTAAAAGTCGTAACAAGG) and ITS2-2043R (GCTGCGTTCTTCATCGATGC) [14] were applied for the amplification of fungal ITS1 region. The PCR amplication, PCR amplicons purification and quantification, and amplicons sequencing were performed according to the method described by Xin et al. [15]. The PCR product was extracted from a 2% agarose gel and purified using an TruSeq DNA PCR-Free Sample Preparation Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturers instructions, and quantified using a Quantus Fluorometer (Promega, Madison, WI, USA). Purified amplicons were pooled in equimolar amounts and paired-end sequenced on an Illumina NovaSeq 6015 platform (Illumina, Inc., San Diego, CA).