3 full race core set with primescript rtase

The full-length cDNA was obtained by Rapid Amplification of cDNA Ends (RACE). 3’-RACE and 5’-RACE cDNA were synthesized using RACE kits (3’-Full RACE Core Set with PrimeScript RTase, 5’-Full RACE Kit, Takara, Japan). The RACE primers(Additional file 1: Table S1) were designed basing on EST sequence of DHN, which acquired by searching “Dehydrin” annotation in L. chinense’s transcriptome database [27 (link)]. All the PCR reactions were carried out in a 50 μL reaction mix according to the PCR protocol (Additional file 1: Table S1). The reaction mix consisted of 5 μL 10 × LA PCR Buffer II (Mg²⁺ Free), 5 μL MgCL₂ (25 mmol·L^− 1), 8 μL dNTP Mixture (2.5 mmol·L^− 1 each), 2 μL of each primer (2.5 mmol·L^− 1 each), 0.5 μL TaKaRa LA Taq (5 U·L^− 1), 2 μL each cDNA, 25.5 μL ddH₂O. PCR products were separated on a 1.5% agarose gel and purified using the DNA gel extraction kit (Transgen Biotech, China). The purified PCR products were cloned into pEASY®-T1 Cloning Vector and transferred into Trans5α Chemically Competent Cells for white-blue plaque selection (Transgen Biotech, China). Positive monoclonal was screened for sequencing (Genscript, China). Finally, the gene full-length was assembled according to the 3′ and 5’sequencing results.

Total RNA of the heart, hepatopancreas, muscle, gill, epididymis, spermatophore and testis were extracted according to the manufacturer's instructions of Trizol reagent (Takara, Dalian, China). All samples were dissolved and homogenized within the Trizol reagent by a homogenizer. The homogenate was sequentially dealt with chloroform, isopropanol and 75% ethanol to make the RNA precipitated. Finally, we dissolved the precipitated RNA from each tissue in 100 μl DEPC-H₂O and measured its concentration by micro-spectrophotometer (Nano-100, Allsheng). All total RNA samples were stored at −80°C.
The PrimeScript® RT reagent Kit (Takara) was used to reverse transcript the total RNA into cDNA. All samples were stored at −20°C for semi-quantitative PCR analysis of KIFC1 mRNA expression in different tissues. In addition, the testis cDNA was used for kifc1 gene cloning. The Smart RACE cDNA Amplification Kit (CloneTech) and the 3′-Full RACE Core Set with PrimeScript™ RTase (Takara) were used for 5′ and 3′ rapid-amplification of cDNA ends (RACE) reverse transcription, respectively.

The molecular clues provided by metagenomic analyses were used to classify sequence reads into a virus family or genus using MEGAN 6. Viral nucleotide (including both viral genome DNAs and viral mRNAs) was isolated using a QIAmp MinElute Virus Spin Kit (Qiagen/United States) from the specimens and used as the template for genome sequencing. Gene-specific primers were designed based on representative identified reads of the novel parvovirus to cover the partial genomes by PCR amplification and Sanger sequencing. The remaining genomic sequences were determined by genome walking (Takara/Japan), 5’-race system version 2.0 combo (Invitrogen/United States) and 3’-Full RACE Core Set with PrimeScript RTase (Takara/Japan).

TRIzol Universal Total RNA Extraction Reagent (cat. no. DP405; Tiangen, Beijing, China) was used to extract RNA from samples. A PrimeScript 1st Strand cDNA Synthesis Kit, 3′-Full RACE Core Set with PrimeScriptRTase, and Smarter RACE 5′/3′ Kit were used to synthesize cDNA (TaKaRa, Dalian, China). TaKaRaTksGflex DNA Polymerase was used for PCR. A TakaRaMiniBEST Agarose Gel DNA Extraction Kit ver. 4.0 was used to recover and purify the PCR product bands. The purified products were treated with a DNA A-Tailing Kit. Ligase from TaKaRa DNA Ligation Kit ver. 2.1 was used for ligation with the T-Vector pMDTM20. The plasmid was heat transferred into Escherichia coli competent cells (JM109), and cells were cultured overnight at 37 °C. Positive single clones were selected and designated CTG0957-PCR-1 and CTG0957-PCR-2. The primers M13-47 and RV-M were used for sequencing.

The partial HIF-1α for Aurelia sp.1 (ASHIF) nucleic acid sequence was obtained from RNA-seq (PRJNA219043) generated by IOCAS. Degenerate primers (CTF653 F0 and CTF653 R0, Table 1) were designed for confirmation of the partial ASHIF gene sequence. A full-length ASHIF transcript was obtained from the total RNA of Aurelia sp.1 medusa using 5′ and 3′ end RACE. For the 5′ end RACE, the cDNA was synthesized via reverse transcription (RT) using the SMARTer RACE cDNA Amplification Kit (Clontech, Mountain View, CA, U.S.A.), and the PCR amplification was performed with the CTF653 R6 and CTF653 R5 primers (Table 1) using Clontech Advantage PCR Kit (Clontech, Mountain View, CA, U.S.A.). Similarly, the cDNA from the 3′ end RACE was synthesized by RT using the 3′-Full RACE Core Set with PrimeScript RTase (Takara, Dalian, China), and the PCR amplification was performed with the CTF653 F2 and CTF653 F3 primers (Table 1) using TaKaRa LA Taq with GC Buffer (Takara, Dalian, China). All products from the 3′ end and 5′ end RACE PCRs were gel-purified using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit (Takara, Dalian, China) and cloned into Escherichia coli using a T-Vector pMD (Takara, Dalian, China). Three positive clones were randomly selected for each gene product and sequenced.

Molecular clues provided by metagenomic analyses were used to classify sequence reads into a virus family or genus using MEGAN 6. Viral RNA was isolated using QIAamp Viral RNA Mini Kit (Qiagen), and cDNA was generated using random primer and Superscript III Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. Gene-specific primers were designed based on representative identified reads of the novel picornavirus while ensuring that they covered the partial genomes by nested-PCR amplification and Sanger sequencing. The remaining genomic sequences were obtained by amplification using a genome-walking kit (Takara, Kusatsu, Japan), and the full 5′- and 3′-end sequences were obtained by repeated amplification using a 5′-RACE system version 2.0 combo (Invitrogen) and 3′-Full RACE Core Set with PrimeScript RTase (Takara) according to the manufacturers’ instructions. All primer sequences were based on the newly obtained reads and newly amplified sequences. All primers used are shown in Supplementary Table S1 online.

Total RNA was isolated from TE1 cells using Trizol reagent kit (Invitrogen, Shanghai, China), according to the manufacturer’s instructions. 3′RACE was performed using the 3′-Full RACE Core Set with PrimeScript Rtase (Code No.6106, Takara, Dalian, China), and 5’RACE was conducted with the SMARTer® RACE 5′/3′ Kit (Cat. No. 634860, Takara, Dalian, China), following the manufacturer’s protocols. All cDNA products were amplified by the TaKaRa Tks Gflex DNA Polymerase (Code No. R060A, Takara, Dalian, China). The RACE PCR products were separated on a 1.5% agarose gel using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (Code No. 9762) and verified by RNA sequencing on ABI Prism 3730XL DNA Analyzer (Life Technologies, CA, USA). The gene-specific primers (5′RACE, 3′RACE) used for the PCR of the RACE analysis are listed in Suppl. Table S1.