Isolation of Viral RNA for Next-Generation Sequencing Protocol

Partial Purification of RNA Virus

1. Place 25 ml of IBV-infected allantoic fluid into a 50 ml Falcon tube and centrifuge for 10 min, 1,150 × g, 4 °C in a benchtop centrifuge.
2. Take the supernatant and layer on top of 10 ml 30 % sucrose in an ultracentrifuge tube. Balance the tubes carefully.
3. Centrifuge for 4 h, 102,400 × g, 4 °C in an ultracentrifuge.
4. Remove the supernatant in layers, careful not to disturb the pellet.
5. Wipe the sides of the tube with tissue and proceed directly to the next stage, RNA extraction.

RNA Extraction

1. Add 1 ml TRIzol reagent directly to the virus pellet, Carefully pipette up and down to mix.
2. Incubate for 5 min at room temperature.
3. Add 200 μl chloroform and mix by shaking for 15 s.
4. Incubate for 3 min at room temperature.
5. Centrifuge in a benchtop centrifuge for 15 min, 4 °C, 12,075 × g.
6. Carefully take the aqueous top layer, which should be clear, and place in a clean 1.5 ml tube.
7. Add 0.5 ml isopropanol and incubate for 10 min at room temperature.
8. Centrifuge for 10 min at 2,100 × g, 4 °C in a benchtop centrifuge.
9. Carefully remove the supernatant without disturbing the pellet.
10. Add 0.75 m 75 % ethanol to the pellet and mix by pipetting.
11. Centrifuge for 10 min, 12,075 × g, 4 °C in a benchtop centrifuge.
12. Remove the supernatant, very carefully, and wipe the sides of the tube with tissue.
13. Air-dry the pellet for 5–10 min.
14. Resuspend the pellet in 25 μl RNAase-free sterile waterand store at either −20 or −80 °C.