Generacer core kit

To determine the genome sequence of a novel virus, specific primers (Table S1) were designed based on its contig sequences obtained in this study and used in RT-PCR for amplification of overlapped viral fragments. One-step RT-PCR was carried out using the PrimeScript One Step RT-PCR Kit (Takara, Japan). RACE (5′ and 3′ termini) was performed using the GeneRacer Core Kit (Invitrogen, USA). PCR amplicons were purified using the Gel Extraction Kit (Biomega, USA) and then cloned using the pEASY-T1 Vector System (TransGen, China). At least five clones of each PCR amplicon were fully sequenced (Sanger), and sequences of all amplicons were assembled to generate complete genome sequences.

A set of specific primers based on the viral contig sequences were designed using the Primer Premier 5 (Premier Biosoft, USA) to amplify overlapping fragments of each of the new C. japonica viruses (Figure S1). The primers are listed in Table S2. One-step reverse transcription-PCR (RT-PCR) assay was carried out using the PrimeScript One-Step RT-PCR Kit (Takara, Japan). Rapid amplification of cDNA ends-PCR (RACE-PCR) assay was conducted using the GeneRacer Core Kit (Invitrogen, USA). PCR assay was done with the 2 × Taq Master Mix Kit (Quick Load) (Novoprotein, China). The PCR amplicons were purified by the Gel Extraction Kit (Biomega, USA) and cloned into the pEASY-T1 Vector (TransGen, China). Sequence of each amplicon was determined from both directions of five clones by a biotechnology company (Tsingke, China). The full-length genome of each virus was assembled from all amplicons of the virus using the de novo assembly algorithm in SeqMan (DNAStar, USA).

Partial sequences encoding Hm-MyD88 and Hm-SARM have been retrieved from the leech H. medicinalis nervous system EST database33 (link). Full length cDNAs were generated by 5′-RACE using GeneRacer Core Kit (Invitrogen). Double stranded cDNAs from leech nervous systems were ligated to adaptors and these templates were used to PCR amplify 5′-RACE fragments using adaptor specific primers and gene-specific primers deduced from the initial fragment sequences. 5′-RACE-PCR were performed using 2.5 units of Platinum Taq polymerase (Invitrogen) in 1.5 mM of MgCl₂. The cycling parameters were: 96°C/30sec; 5 cycles at 96°C/10sec and 72°C/4 min; 5 cycles at 96°C/10sec and 70°C/4 min; 25 cycles at 96°C/10sec and 68°C/4 min. All PCR products were cloned using pGemT-easy vector (according to the protocol provided by the manufacturer) and transformed into competent Escherichia coli JM 109 cells (Promega). Plasmids DNA were sequenced with a FM13/RM13 sequencing kit (Applied Biosystems) according to the manufacturer's instructions.