Isolation and Purification of IBDV dsRNA

Genomic dsRNA was extracted from preparations of sucrose gradient-purified IBDV as previously described [16] (link). Briefly, purified IBDV preparations were incubated with 1% SDS for 3 min at 100°C followed by treatment with proteinase K (2 mg/ml, 1 h, 37°C). The dsRNA was then extracted with TriZol (Invitrogen) and purified using silica-based mini-spin columns (Quiagen). Isolated dsRNA samples were treated with 50 units/µg of dsRNA of RNase T1 (Roche), specifically digesting ssRNA, at 37°C for 30 min to eliminate potentially contaminating ssRNA traces, and then subjected to a second round of purification on mini-spin columns. dsRNA concentrations were determined using a Nanodrop spectrophotometer (Thermo scientific). The effectiveness of the RNase T1 was tested on irrelevant ssRNA samples. For control experiments, RNase T1-treated purified dsRNA was treated with RNase III (Invitrogen), specifically digesting dsRNA, to eliminate long dsRNA molecules. RNA samples were incubated for 1 h at 37°C with 1 U of RNase III, and then purified as described above.