title: Metatranscriptomic analysis of sub-acute ruminal acidosis in beef cattle
design: Approximately 0.25 grams of each sample underwent RNA extraction using the RNeasy PowerMicrobiome Kit (Qiagen). The RNeasy PowerMicrobiome kit utilizes a cell lysis protocol that is similar to the PowerSoil DNA isolation kit, which relies on the addition of each sample to a beaded tube in combination with Lysis buffer (Solution PM1/- mercaptoethanol), and were subsequently vortexed within the Disruptor Genie for 10 minutes. All RNA extracts were quantified using the Qubit RNA High Sensitivity Kit (Invitrogen, Carlsbad, CA, USA) to confirm complete DNase treatment of the RNA extracts (DNA concentration <0.05 ng/uL). Subsequently, approximately 100 ng of extracted RNA was subjected to NuGEN Ovation (NuGEN Technologies, San Carlos, CA, USA) double stranded cDNA synthesis and metatranscriptome library preparation. Quality of the final library was assessed using a high sensitivity bioanalyzer chip (Agilent). The same library quantification, pooling, and purification methods were used as described above. Purified metatranscriptome libraries underwent sequencing on the Illumina HiSeq4000 following a 2 x 150bp index run