Multina mce 202 with dna 2500 kit

RNA was reverse-transcribed by using 4× VirDect 1-step RT-qPCR Master Mix with random primers (Topgen Biotech., Taiwan) to generate cDNA. To enable a fast-sequencing approach, amplifications were performed using 10 ng cDNA with the TopPLUS PCR Master Mix (Topgen Biotech., Taiwan) and specific target primer pairs with a working concentration of 250 nM and an Applied Biosystems 9700 Thermal Cycler (Applied Biosystems, United States) according to the manufacturer’s instructions. The thermal cycling program was as follows: 95°C for 3 min, 32 cycles of 95°C for 15 s, 60°C for 20 s, and 72°C for 40 s, and a final extension at 72°C for 2 min. The amplified products were purified with VAHTS DNA Clean Beads (Vazyme Biotech., China), analyzed using a MultiNA MCE 202 with DNA 2500 Kit (Shimadzu, Japan) to check the target amplicon length and quantity. Sanger sequencing was then performed according to the manufacturer’s protocol to confirm variants and indel regions.

The RNA was reverse-transcribed by using 4x VirDect 1-step RT–qPCR Master Mix with random primers (Topgen Biotechnology Co., Kaohsiung, Taiwan) to create cDNA. To enable a fast-sequencing approach, amplifications were performed using 10 ng cDNA with TopPLUS PCR Master Mix (Topgen Biotechnology Co., Kaohsiung, Taiwan) and specific target primer pairs (Supplementary Table 1) with a working concentration of 250 nM on an Applied Biosystems 9700 Thermal Cycler (Applied Biosystems, USA) according to the manufacturer's instructions. The thermal cycling program used a protocol of 95°C for 3 min, 32 cycles of 95°C for 15 s, 60°C 20 s 72°C for 40 s, and a final extension of 72°C for 2 min. The amplified products were purified with VAHTS DNA Clean Beads (Vazyme Biotech Co., Nanjing, China) and were analyzed on the MultiNA MCE 202 with DNA 2500 Kit (SHIMADZU Co., Kyoto, Japan) to check the target amplicon length and quantity, and then the amplified products were used for Sanger sequencing according to manufacturer's protocol to confirm the single nucleotide variations (SNVs) and indel regions, respectively.