Virus inactivation and RNA extraction with TRIzol.

After removal of the supernatant, monolayers of filovirus-infected or mock-infected cells were treated with 1 ml TRIzol (Ambion), incubated for 10 min at room temperature, and removed from the BSL-4 facility. A 200-μl volume of chloroform per 1 ml of TRIzol used was added to each sample, and the samples were incubated at room-temperature for 5 min, after which they were centrifuged at 12,000 × g for 20 min. The aqueous phase was removed to fresh nuclease-free 1.5-ml tubes, to which 15 μg of linear acrylamide (Ambion) and 1.0 ml of 2-propanol per ml of TRIzol used was added. Samples were incubated at −20°C overnight to precipitate RNA. Following incubation, the samples were centrifuged at maximum speed (16,900 × g) at 4°C for 30 min and were then washed three times with 80% ethanol (each wash was followed by centrifugation at maximum speed for 30 min at 4°C). Precipitated RNA pellets were allowed to air dry for ∼5 min before being resuspended in nuclease-free water. Resuspended total RNA was quantified and was assessed for purity by a NanoDrop system (Thermo Fisher).