5 full race core set kit

The 5′ RACE experiment was performed using a 5-Full RACE Core Set Kit (TAKARA, Japan) in accordance with the manufacturer’s instructions. Fifteen microliters of ligated cDNA was then used as a template for nested PCR using KOD FX Neo polymerase (TOYOBO, Japan) with two sets of primers, the outside set was sense primer_1 and antisense primer_1, and the inside set is sense primer_2 and antisense primer_2. The PCR reaction mixture was incubated for 2 min at 94 °C followed by 30 amplification cycles, comprising denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s and extension at 68 °C for 30 s. The reaction was extended for another 10 min at 68 °C to ensure full extension. Primers used in this study are listed in Supplementary Table S2.

To determine the 5'-end of the SaV genomic RNA, RACE (Rapid-Amplification of cDNA Ends) was performed with the 5'-Full RACE Core Set Kit (Takara Bio Inc., Ohtsu, Japan). The first cDNA strand was synthesized through reverse transcription from target mRNA using 5' end-phosphorylated RT Primer (5’-SV-PR, Table 1), after which it was treated with RNAse H to remove hybrid RNA and with RNA Ligase to form circularized single-strand cDNA or concatemers. To amplify the product, the first PCR reaction was performed using 5’-SV-F1 and 5’-SV-R1 primers under the following conditions: 94°C for 3 min, followed by 25 cycles each of 94°C for 30 sec, 56°C for 30 sec and 72°C for 5 min. Then, the second PCR reaction was conducted with 5’-SV-F2 and 5’-SV-R2 primers through 30 cycles of 3-step cycling (94°C for 30 sec, 56°C for 30 sec and 72°C for 5min).
To attain the exact sequence for the 3'-end of the SaV genomic RNA, cDNA was synthesized using RT reaction performed with 3'-end poly A tail-based 3’-Oligo (dT)-anchor primer (Table 1). The second PCR reaction was conducted using the SV-10F and 3’-anchor-R primers (Table 1) under the following conditions: 30 cycles of 3-step cycling (98°C for 10 sec, 56°C for 30 sec and 72°C for 1min) and 72°C for 7min.