title: Rumen metatranscriptomics
design: Total RNA was extracted from rumen fluid and culture fluid using PowerSoil Total RNA Isolation Kit (MoBio, USA) following the manufacturer's instructions. The quality and quantity of the extracted RNA were assessed using 1.5% agarose gel electrophoresis and a UV spectrophotometer. The RNA integrity (RIN  7) was measured using an Agilent 2100 Bioanalyzer (Agilent, USA). Ribosomal RNA was removed from total RNA using a hybridization-based method with magnetic beads, according to the procedures indicated in Ribo-Zero Plus Microbiome kit (Illumina, USA). The TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, USA) was used for reverse transcription and construction of the metatranscriptomic sequencing library.