Specific primer pairs were designed at appropriate positions of the putative viral contigs using the CLC Genomics Workbench 11 and the Primer Premier 5.0 (Premier Biosoft, Palo Alto, CA, USA) to amplify overlapping fragments (Supplementary Table S4 ). Then, a one-step reverse transcription-PCR (RT-PCR) assay was conducted with a PrimeScript kit (Takara, Tokyo, Japan), and viral genomic terminal sequences were determined using commercial 5′ and 3′ RACE (rapid amplification of cDNA ends) kits (Invitrogen, Waltham, MA, USA). The PCR products were gel-purified using the Gel Extraction Kit (OMEGA Bio-Tec Inc., Doraville, GA, USA) and cloned on pEASY-T1 vectors (TransGen, Beijing, China) using competent cells. Five clones of each amplicon were fully sequenced with primer in both directions (Tsingke, Chengdu, China), and the output sequences were de novo assembled in the SeqMan program (DNAStar, Madison, WI, USA).
5 and 3 race
Manufactured by Thermo Fisher Scientific
Sourced in United States
5' and 3' RACE is a laboratory technique used to determine the complete sequence of RNA transcripts by identifying the 5' and 3' ends of the RNA molecules. This method allows for the amplification and analysis of the full-length cDNA sequences from limited amounts of total RNA.
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2 protocols using 5 and 3 race
1
Complete Viral Genome Sequencing
2
Viral Genome Sequencing and Assembly
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Specific primer pairs were designed at appropriate positions of the viral contigs to amplify overlapping fragments using the CLC Genomics Workbench 11 and the Primer Premier 5.0 (Premier Biosoft, Palo Alto, CA, USA). Thereafter, a one-step reverse transcription-PCR (RT-PCR) assay was conducted with a PrimeScript kit (Takara, Tokyo, Japan), and Viral genomic terminal sequences were determined using commercial 5′ and 3′ RACE (rapid amplification of cDNA ends) kits (Invitrogen, Waltham, MA, USA).
The PCR products were gel-purified using the Gel Extraction Kit (OMEGA Bio-Tec Inc., Doraville, GA, USA) and cloned on pEASY-T1 Vectors (TransGen, Beijing, China) using competent cells. Five clones of each amplicon were fully sequenced with primer in both directions (Tsingke, Chengdu, China), and the output sequences were de novo assembled in the SeqMan program (DNAStar, Madison, WI, USA).
The PCR products were gel-purified using the Gel Extraction Kit (OMEGA Bio-Tec Inc., Doraville, GA, USA) and cloned on pEASY-T1 Vectors (TransGen, Beijing, China) using competent cells. Five clones of each amplicon were fully sequenced with primer in both directions (Tsingke, Chengdu, China), and the output sequences were de novo assembled in the SeqMan program (DNAStar, Madison, WI, USA).
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