Miseq pe300 platform

Genomic DNA was extracted from the intestinal bacteria using a bacterial DNA isolation kit (Foregene, Chengdu, China) according to the manufacturer’s instructions. The extracted DNA was checked on 1% agarose gel, and DNA concentration and purity were determined with NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, DE, USA). After the extraction, DNA fragments from the samples were amplified using specific primers: 338F: 5′-ACTCCTACGGGAGGCAGCAG-3′, 806R: 5′-GGACTACHVGGGTWTCTAAT-3′ [61 (link)]. The PCR product was detected using 2% agarose gel, purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified using Quantus™ Fluorometer (Promega, Madison, WI, USA). V3–V4 amplicon library was constructed using TruSeqTM DNA Sample Prep Kit (Illumina, San Diego, CA, USA). High-throughput sequencing was performed in a paired-end model using the Illumina MiSeq PE300 platform (Illumina, San Diego, CA, USA) by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China).

Approximately 3.3 g of FCB samples were weighed, and then transferred into 50 mL sterile centrifuge tubes containing 15 mL of 1 × PBS buffer (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China). The mixture was shaken for six minutes and filtered through four layers of sterile gauze. All filtrates were centrifugated at 12,000 rpm for 26 min to collect fungal strains for total DNA extraction. The fungal DNA was extracted from FCB samples by using an EZNA^® soil DNA kit (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer protocols. We amplified the internal transcribed spacer 2 (ITS2) sequences by using the universal primers ITS3/ITS4 [20 ]. The amplification conditions were as follows. Initial denaturation at 95 °C for 3 min; 35 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, elongation at 72 °C for 45 s, followed by a final extension at 72 °C for 10 min. The DNA products were checked for quality through 2% agarose gel electrophoresis. The purified DNA amplicons were pooled in equimolar quantities, and sequenced through Illumina Miseq PE300 platform (Illumina, San Diego, CA, USA).

Microbial DNA was extracted from rumen fluid samples (30 samples in total) using PowerSoil DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA) following the manufacturer’s guidelines. The concentration and purity of the extracted DNA were measured by a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States). The V3–V4 region of the bacterial 16S rRNA gene was amplified using primer 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) (Takahashi et al., 2014 (link)). PCR amplification proceeded through an initial denaturing step at 94°C for 5 min; 28 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 60 s; and a final extension at 72°C for 7 min using 25-μl reaction volumes, containing 12.5 μl 2 × Taq PCR MasterMix, 3 μl BSA (2 ng/μl), 1 μl forward primer (5 μM), 1 μl reverse primer (5 μM), 2 μl template DNA, and 5.5 μl ddH2O. The PCR products were detected by 2% agarose gel electrophoresis, purified with the Agencourt AMPure XP kit (Beckman Coulter, La Brea, CA, United States) according to the manufacturer’s instructions, and quantified by QuantiFluor-ST (Promega, Madison, WI, United States). Purified amplicons were sequenced on an Illumina MiSeq-PE300 platform (Illumina Inc., San Diego, CA, United States), generating 2 × 300 bp paired-end reads.