T4 rna ligase

The 5′ and 3′ end regions were amplified as previous methods described by Potgieter et al. (2009) (link), with minor modifications. The extracted dsRNA was first subjected to gel purification following the AxyPrep™ Nucleic Acid Purification Kit's instructions (AXYGEN, Suzhou, China). The terminal sequences of the purified dsRNA were ligated with the PC3-T7 loop (5′-p-GGATCCCGGGAATTCGGTAATACGACTCACTATATTTTTATAGTGAGTCGTATTA-OH-3′) by mixing and establishing a system comprising BSA, Recombinant RNase Inhibitor, T4 RNA ligase 40 U/μL, 10 ​× ​T4 RNA buffer (Takara, Dalian, China), 50% PEG 6000 and supplemented with ddH₂O. The mixture was incubated at 4 ​°C for 18 ​h. Following the denaturation of the purified ligated dsRNA with Dimethyl sulfoxide, the denaturated dsRNA was thereafter cloned via RT-PCR. The resultant cDNA was subsequently amplified with 5′-GTGGCAAAACACCCGAAGAC-3′ and 5′-CTCCGAGCGTAGTTTGGGTT-3′ for the 3′ and 5′ ends, respectively, as the forward primers, and a complementary sequence to PC3-T7 loop, PC2 (5′-p-CCGAATTCCCGGGATCC-3′) as the reverse primer. The amplicons obtained were subsequently subjected to gel purification (Takara, Dalian, China), cloned in a vector pMD18-T (Takara, Dalian, China), and sequenced. At least four-time repetitions were performed during the 3′ and 5′ end amplification.

5′RACE was performed according to the method described in Bensing et al. (1996) (link). Briefly, 6 μg of total RNA was treated with 75 U of tobacco pyrophosphatase (TAP; Nippon Gene) at 37°C for 30 min in the presence of 20 U of RNaseOUT. The TAP-treated and -untreated RNA samples were mixed with the RNA oligonucleotide (5′-AUAUGCGCG AAUUCCUGUAGCUAGAAGAAA-3′) and ligated by 40 U of T4 RNA ligase (TAKARA Bio) at 16°C overnight. The ligated RNA samples were mixed with 1 pmol of gene-specific primer traJ-R2 (5′-TCTCTTCGATCTTCGCCAGC-3′) and reverse transcribed by 100 U of SuperScriptIII at 50°C for 60 min in the presence of 20 U of RNaseOUT. The cDNA fragment spanning the ligated RNA oligonucleotide and the 5′ end of traJ transcript was amplified by KOD-Plus high-fidelity DNA polymerase (TOYOBO) using primers Oligo-F1 (5′-TATGCGCGAATTCCTGTAGC-3′) and traJ-R. The amplified fragment was cloned into HincII-digested pBluescript II SK(-) vector (Stratagene), and the inserts from several clones were sequenced using M13 primers.

Total RNA was extracted from three samples as previously described [46 (link)] and used for sRNA and qRT-PCR assay. RNA quality and concentration were determined via 1.2% agarose gel electrophoresis and NanoDrop 2000c spectrophotometer (NanoDrop, Wilmington, DE, USA), respectively.
For construction and sequencing of the sRNA libraries, two replicates were performed for each sample. Firstly, sRNAs were isolated from total RNA by polyacrylamide gel electrophoresis (PAGE). Next, the isolated sRNAs were added to a 5′ RNA adaptor and a 3′ RNA adaptor by using T4 RNA ligase (TaKaRa, Dalian, China). Then, sRNAs with added 5′ and 3′ RNA adaptors were reverse transcribed into single-stranded cDNA using RT-PCR. Follows, the single-stranded cDNA was further synthesized into double-stranded cDNA by PCR amplification using adapter primers. Finally, the PCR product was purified and subjected to high-throughput sequencing by using the Illumina SE50 system at Biomarker Technologies Co., Ltd. (Beijing, China).

The terminal sequences of dsRNAs were determined by the following method. Pre-denatured dsRNAs in DMSO (90%, 65°C) were ligated at their 3'-ends with a 5'-phosphorylated oligodeoxynucleotide, 3'-rapid amplification of cDNA ends (RACE) adaptor (5'-CAATACCTTCTGACCATGCAGTGACAGTCAGCATG-3') using T4 RNA ligase (Takara) at 16°C for 16 h. Ligated DNA-RNA strands were DMSO-denatured in the presence of the oligonucleotide 3'-RACE-1st (5'-CATGCTGACTGTCACTGCAT-3') and used as templates for cDNA synthesis. The resulting cDNA was further amplified by secondary PCR with 3'-RACE-2nd (5'-TGCATGGTCAGAAGGTATTG-3'), and gene-specific primers were designed for the desired targets.
The obtained cDNA fragments were cloned into the pGEM-Teasy (Promega, Madison, WI, United States) or pCR-Blunt cloning vectors (Thermo Fisher, Waltham, MA, United States). These were used for transformation of E. coli strain DH5α or TOP10 for Sanger sequencing analyses. Plasmid clones were used for BigDye sequencing (ABI, ThermoFisher, Carlsbad, CA, United States) on a 3,100-Avant sequencer (ABI/Hitachi, Foster City, CA, United States) following the manufacturer’s instructions.

Viral RNA was extracted from purified GCRV-GZ1208 using Trizol^® Reagent (see above) and full-length amplification of cDNA (FLAC) was used to amplify the entire genome of GZ1208 as described previously [41 (link)]. In brief, an anchor primer (5′-p-GACCTCTGAGGATTCTAAAC/iSp9/TCCAGTTTAGAATCC-OH-3′) possessed a C9 spacer between two complementary halves and a phosphorylated 5′ terminus. The anchor primer was ligated to the 3′ ends of the dsRNA segments using T4 RNA ligase (Takara, Tokyo, Japan). Then, the reaction mixtures were purified using a commercial RNA clean up kit (Qiagen, Hilden, Germany) and used as templates for first-strand cDNA synthesis using an AMV Reverse Transcription System (Takara, Dalian, China) and PCR amplification using a complementary primer (5′-GAGGGATCCAGTTTAGAATCCTCAGAGGTC-3′). PCR products were separated on a 1.2% agarose gel, and visible bands were purified using a Silica Bead DNA Gel Extraction kit (Fermentas, Waltham, MA, USA) and cloned into a pMD18-T vector (Takara, Dalian, China). Positive clones were sent to Sangon Biotech (Shanghai, China) for sequence analysis.