Pjet 1.2 blunt end cloning vector

Reverse transcription-polymerase chain reaction (RT-PCR) was performed to amplify the viral RNA for sequencing and phylogenetic analysis. Viral RNA was extracted using TRIzol^TM Reagent (Thermo Fisher Scientific, USA) and amplified in one reaction with reverse transcriptase M-MLV (RNase H-), recombinant RNase inhibitor (TaKaRa, Japan) and the oligonucleotide universal primer 5’-AGCAAAAGCAGG-3’. The genome was amplified by PCR using the Platinum^TM Pfx DNA polymerase kit (Thermo Fisher Scientific, USA) with a series of primers[22 (link)]. The products were then purified using an agarose gel DNA purification kit (TaKaRa, Japan). To sequence the genes, the products were cloned into the pJET 1.2 blunt-end cloning vector (Thermo Fisher Scientific, USA) and transformed into competent DH5α (Tiangen Biotech Beijing Co., Ltd.). At least three clones per gene were sequenced by Sanger sequencing (Thermo Fisher Scientific, USA).

RNA from ANA positive hybridomas was extracted using TRI Reagent (Sigma) followed by cDNA synthesis (GoScript Reverse Transcriptase) according to the manufacturer’s protocol and using primers as specified in Table S5 in Supplementary Material. Amplification of the heavy and light chain V-regions was performed using Vent polymerase (New England Biolabs) and, subsequently, the Vh and Vl regions were ligated into the pJet1.2 blunt end cloning vector (Thermo Fisher Scientific). For sequencing, plasmids were sent to Microsynth (Balgach, Switzerland). Resulting sequences were inspected using DNASTAR and aligned to the germline heavy and light chain sequences of the international ImMunoGeneTics information system^® (http://imgt.org).

A total of 1.5 µg total RNA extracted from inoculated N. clevelandii was subjected to cDNA synthesis using Maxima H Minus Reverse Transcriptase and random hexamers (Thermo Scientific™, Waltham, MA, USA) following the manufacturer’s protocol. The cDNAs were applied as a template in PCR for amplification of the genomic region of ToBRFV from nucleotides 1482-6393 (numbers are based on the GenBank accession number NC028478) in three segments using the primer pairs of ToBRFV-1482-s/ToBRFV-4750-as, ToBRFV-KpnI-4388-s/ToBRFV-HindIII-6153-as, and ToBRFV-CP-Eco47-s/ToBRFV-HindIII-3UTR-as, and Phusion™ High-Fidelity DNA Polymerase kit (Thermo Scientific, Waltham, MA, USA). PCR products were gel extracted and were inserted into pJET1.2 Blunt end cloning vector (Thermo Scientific, Waltham, MA, USA) in three steps through compatible DNA restriction sites available in the ToBRFV genome, the vector, and the primers. The final recombinant plasmid was named pJET-ToBRFV-1482-6393. The recombinant plasmids were sent to Macrogen (Macrogen Europe, Amsterdam, The Netherlands) for sequencing, and each nucleotide was sequenced at least twice. This plasmid was used as a template for the preparation of the plasmid standard curve in RT-qPCR and in vitro transcription.