RNA-Seq of rumen fluid: adult male  ovis aries
Genomic DNA was extracted from rumen fluid samples using SDS technique before assessing the DNA's purity and concentration. The V3-V4 regions of 16S rRNA genes were then amplified by PCR using specific primers under the following conditions: initial denaturation for 1 min at 98 C, followed by 30 cycles, each with denaturation for 10 s at 98 C, annealing for 30 s at 50 C and extension for 60 s at 72 C. The PCR ended with a 5-min final extension at 72 C. AxyPrep DNA Gel Extraction Kit (AXYGEN, USA) and QuantiFluor-ST (Promega, USA) was then used to respectively purify and quantify the PCR products as specified by the manufacturer. This was followed by the preparation of a sequencing library using an Illumina NextUltraDNA Library Prep Kit (NEB, USA), with both Qubit@ 2.0 Fluorometer and Agilent Bioanalyzer 2100 systems used to assess the quality of the library. Eventually, sequencing was performed on an Illumina MiSeq/HiSeq2500 platform to generate 250bp/300bp paired-end reads.