Buffer rlt 1 β mercaptoethanol

Viral RNA was isolated from mouse serum and Huh-7.5 supernatant using the ZR Viral RNA Kit (Zymo) according to manufacturer's instructions. Total RNA was extracted from Huh7.5 cell pellets and mouse tissues (spleen, brain, liver and kidney) using the RNeasy Mini Kit (Qiagen) following manufacturer's instructions. Isolated tissues (20–30 mg) were suspended in Buffer RLT-1% β-mercaptoethanol (Qiagen), lysed using TissueLyser (Qiagen; 20 cycles per s for 2 min, 1 min wait, 20 cycles per s for 2 min) and centrifuged at high-speed. The resulting supernatant was used for extracting viral RNA.

At the indicated time points, 200 µl of blood was collected through submandibular bleeding in a 1.5-ml collection tube. Serum was then separated from blood cells by centrifugation (10 min, 3,500 rpm). Viral RNA was isolated from mouse serum using a ZR viral RNA kit (Zymo, Irvine, CA) according to the manufacturer’s instructions. At the indicated endpoints, mice were euthanized via exsanguination under ketamine/xylazine anesthesia. Spleen, brain, liver, kidney, and thymus tissues were collected, placed into RNAlater (Ambion, Foster City, CA) solution, and maintained overnight at 4°C. The isolated tissues (20 to 30 mg) were then resuspended in buffer RLT–1% β-mercaptoethanol (Qiagen, Hilden, Germany), lysed using a TissueLyser (Qiagen, Hilden, Germany) (20 cycles/s for 2 min, 1 min wait, 20 cycles/s for 2 min), and centrifuged at high speed (10,000 rpm) for 10 min. Total RNA was then extracted from the resulting supernatant using an RNeasy minikit (Qiagen, Hilden, Germany) following the manufacturer’s instructions.