GSM5005578: T3D infected L929 murine fibroblasts replicate 1; Mus musculus; Mammalian orthoreovirus 3 Dearing; RNA-Seq
Infected cell pellets were thawed, incubated with 0.14% deoxycholate for 30 min on ice, then sonicated in the presence of Vertrel XF to release virus particles from cells. Virions and TC particles were separated by ultracentrifugation at 25,000 × g in a 1.2-1.4 g/cm3 cesium chloride density gradient. Mock virions and TC particles were collected by aligning gradients containing virions and TC particles next to mock gradients, marking the expected position of virions and TC particles onto the mock gradient, aspirating liquid above the expected position, and transferring 250 µL of the gradient from the expected position of virions and TC particles into clean Eppendorf tubes. Mock preparations and complete virus particles and TC particles were collected and dialyzed in virion storage buffer (150 mM NaCl, 15 mM MgCl2, 10 mM Tris-HCl, pH 7.4). Purified reovirus particles were treated with 1 U/µL benzonase (Sigma) for 1 h at 37°C to degrade extra-particle nucleic acids. RNA content was analyzed by Bioanalyzer (Agilent) before libraries were prepared. Libraries were prepared using NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs), then Illumina-ready libraries were sequenced by 150 base pair paired-end sequencing on the NovaSeq 6000 Sequencing System (Illumina)