RNA-Seq of Rotavirus A: young pigs
About 0.5-1.0 g of stool samples positive for the RVA genes were added to 10 mL of PBS and vortexed until well blended. The sus-pension was centrifuged at 10,000 x g for 3 min, and the supernatant was filtered through a 0.45 m membrane to remove unpelleted bacterial-sized substances. The fil-trate was concentrated using Amicon Ultra-15 centrifuge filters (Merck Millipore, Darmstadt, Germany) according to the manufacturer's instructions. The subsequent filtrate was used as material for RNA extraction with the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturers instructions. Then, viral dsRNA in extracted total RNAs were purified using the ISOVIRUS II kit (Nippon Gene, Tokyo, Japan) according to the manufacturers instructions. The purified dsRNA was used for cDNA synthesis. Double-stranded cDNA was synthesized using a Prime Script Double Strand cDNA Synthesis Kit (Takara Biotechnology Co., Ltd.) according to the manufacturers instructions. The cDNA libraries were prepared using Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA) according to the manufacturers instruction and subsequently subjected to whole-genome sequencing on the Illumina HiSeq X Ten