TLPs and DLPs were purified as previously described [26 (link)]. For TLP and DLP production, MA104 cells were grown in 850 cm2 roller bottles (Corning), and confluent monolayers were infected with rhesus rotavirus (RRV, G3 serotype) at MOI (multiplicity of infection) of 0.1 focus-forming unit (FFU)/cell in M199 medium supplemented with 1 mg/mL porcine pancreatic trypsin (Worthington Biochemical). Cell culture medium was collected 24–36 h post infection, when cell adherence was <5%. TLPs and DLPs were purified by freeze-thawing, ultracentrifugation, Freon-113 extraction, and separation on a cesium chloride gradient. TLPs were de-salted with a 5 ml Zeba spin column (Thermo Fisher) into 20 mM Tris pH 8.0, 100 mM NaCl, and 1 mM CaCl2 (TNC). DLPs were de-salted into 20 mM HEPES pH 7.5, 100 mM NaCl (HN).
For data collection of transcribing particles [20 (link)], 21 μl of TLPs (3.5 mg / ml) or DLPs (2.7 mg / ml) were added to a final reaction volume of 30 μl containing 150 mM NaCl; 9 mM MgCl2; 4 mM adenosine-5’-triphosphate (ATP) (New England Biolabs); 2 mM each of guanosine-5’-triphosphate (GTP), cytidine-5’-triphosphate (CTP), and uridine-5’-triphosphate (UTP) (New England Biolabs); and 640 μM S-adenosylmethionine (SAM) (New England Biolabs). The reaction was incubated for 5 min at 37 °C and placed on ice until grids were prepared.
For data collection of transcribing particles [20 (link)], 21 μl of TLPs (3.5 mg / ml) or DLPs (2.7 mg / ml) were added to a final reaction volume of 30 μl containing 150 mM NaCl; 9 mM MgCl2; 4 mM adenosine-5’-triphosphate (ATP) (New England Biolabs); 2 mM each of guanosine-5’-triphosphate (GTP), cytidine-5’-triphosphate (CTP), and uridine-5’-triphosphate (UTP) (New England Biolabs); and 640 μM S-adenosylmethionine (SAM) (New England Biolabs). The reaction was incubated for 5 min at 37 °C and placed on ice until grids were prepared.