RNA Seq of sheep:rumen fluid CK5
24 healthy 6-month-old small-tailed Han sheep  Hu sheep hybrid generation rams (24.4  0.59 kg) were selected to graze on pastures of Medicago sativa and Festuca arundinacea Schreb (1:1) supplemented with 0, 0.83, 1.67, and 3.33 g/kg BW of CS, respectively. Microbial DNA was extracted from rumen fluid using a stool DNA kit, and DNA concentration was detected spectrophotometrically (ND-2000, NanoDrop Technologies, Wilmington, DE, United States), and DNA quality was checked using 1% agarose gel electrophoresis. The V3-V4 region of the bacterial 16S rRNA gene was amplified using primers 338F (5-ACTCCTACGGGAGGCAGCAG-3) and 806R (5-GGACTACNNGGGTATCTAAT-3). Fungi utilized the primers ITS1-F (5-CTTGGTCATTTAGAGGAAGTAA-3) and ITS2-R (5-GCTGCGTTCTTCATCGATGC-3) to sequence the hypervariable region ITS1 of the ribosomal RNA. PCR products were purified by Agencourt AMPure XP kit (Agencourt, Cat. No. A63882, Beckmancoulter, Suzhou, China) and concentration and quality were detected using Caliper LabChip GX Touch HT (PerkinElmer). Purified PCR products were subjected to library construction using the NEB Next Ultra II DNA Library Prep Kit, followed by high-throughput sequencing via the Illumina MiSeq PE250 platform (Beijing Allwegene Technology Co., Ltd.). After the raw data were down-loaded, primers and sample identification sequences were removed, followed by quality filtering and assembly processing, to generate raw tags. High quality sequences of bacteria and fungi were clustered using the UPARSE algorithm, and sequences were clustered into operational taxonomic units (OTUs) with a sequence similarity threshold of 97% 20. Using the BLAST algorithm, the representative sequences of bacterial OTUs were aligned against the Silva138 database (http://www.arb-silva.de) to obtain the corresponding taxonomic information for each OTU 21. The representative sequences of fungal OTUs were subjected to a BLAST search against the Unite database