Hiscribe t7 fast high yield rna synthesis kit

An IAV (H1N1) subtype-specific crRNA was designed (Table 1). First, polymerase acidic protein (PA), polymerase basic protein 1 (PB1), nucleoprotein (NP), polymerase basic protein 2 (PB2), and non-structural protein (NS) sequences of influenza A virus PR8 strain were aligned using MEGA7. A nucleic acid fragment of 28 bases was designed as a crRNA. The crRNA was prepared as in the previous study (19 (link)). The product was transcripted using the HiScribe T7 Fast High Yield RNA Synthesis Kit (NEB) and incubated overnight at 37°C. Finally, crRNA was purified using Beckman Coulter RNA clean XP (Beckman Coulter, USA) at a ratio of 1:1.8.

RT-PCR primers (Supplementary Table S1) targeting SARS-CoV-2 HV69-70del and N501Y mutations were designed using the Primer-BLAST tool from the NCBI website. To design the specific crRNAs of the SARS-CoV-2 variant, the genome sequence was downloaded and compared from GISAID; crRNAs were designed to nucleic acid fragments of 28 bp. The crRNA transcripts were prepared by annealing with T7-crRNA-F and crRNA-R. crRNAs were prepared by first synthesizing DNA with a T7 promoter sequence. The crRNA DNA was then annealed to a short T7 sequence with the HiScribe T7 Fast High Yield RNA Synthesis Kit (NEB) and incubated overnight with T7 polymerases at 37°C. Finally, the RNA clean XP volume (Beckman Coulter) was used for crRNA purification at a ratio of 1:1.8. All crRNA sequences used in this study are available in Supplementary Table S2.