Poly a polymerase

Sequencing of the 5’ and 3’ termini of the viral genome was performed using a 5’/3’ RACE kit (Roche) following the manufacturer’s protocol. All primers used are described in Table 2. To obtain the 5’ end sequence of the ZIKV genome 5’ RACE was performed. Briefly, 1 μg total RNA was extracted from ZIKV-infected Vero E6 cells using a Direct-zol RNA mini kit and reverse transcribed using the ZIKV specific primer, SP1. The synthesized cDNA was purified using the illustra GFX PCR DNA and Gel Band Purification kit (GE Healthcare) according to the manufacturer’s instructions. This was prior to polyadenylation at the 3’ end and amplification using the PCR anchor primer and a ZIKV specific primer (5’ PCR). 3’ RACE was carried out to obtain the 3’ end sequence using 1 μg total RNA extracted from ZIKV infected Vero cells which was polyadenylated at the 3’ end using Poly(A) polymerase (New England Biolabs) following the manufacturer’s guidelines. cDNA synthesis was performed by reverse transcribing the RNA using the oligo (dT) anchor primer. Amplification of the cDNA was achieved by using the PCR anchor primer and a ZIKV specific primer (3’ PCR). The PCR cycling conditions were 95°C for 2 min then 35 cycles of 95°C 20 sec, 56°C (5’ RACE) or 68°C (3’ RACE) for 10 sec, 70°C for 15 sec and 70°C for 7 min.

Detection and quantitation by RT-qPCR of the small RNAs derived from the dsRNAs was performed as described previously [22 (link)]. In this section, we quantified the 6125-vsiRNAs derived from the sprayed dsRNA, as a product of the RNAi machinery from the plant cell. Briefly, the RNA extracted from the plant samples was polyadenylated with the poly A polymerase (NEB, Ipswich, MA, USA) and reverse transcribed using the primer polyT (5′-GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTVN-3′), as described by Shi and Chiang [66 (link)] and the High-Capacity cDNA Reverse Transcription Kit. The cDNA was then used to detect by qPCR the 6125-vsiRNA using the primer CG-6125 (5′- GCTAGGGCTGAGATAGATAATT-3′) and the universal reverse primer (URP) (5′-GCGAGCACAGAATTAATACGAC-3′). Reaction and cycling conditions were described previously [22 (link)]. For the reference with an endogenous plant siRNA, we used the primer CUC5.8S based on the 5.8S rRNA of C. sativus (5′-CTTGGTGTGAATTGCAGGATC-3′) [22 (link)]. Six biological replicas were included in each condition and the experiment was repeated twice. Each qPCR (technical repetition), including those for the 5.8S as internal control, was repeated three times. The specificity of the amplicons obtained was checked as above and the relative expressions of the vsiRNAs were calculated as described previously.

Libraries of cDNA corresponding to 5′-monophosphorylated ends present before and after incubation with RNase E were constructed and sequenced as a service provided by vertis Biotechnologie AG (Germany). As described previously (36 (link)), the 5′-sequencing adaptor was ligated to transcripts prior to fragmentation, thereby allowing the 5′ ends of both long and short transcripts to be cloned. RNA was fragmented using a Bioruptor^® Next Gen UCD-300™ sonication system (Diagenode), then tailed at the 3′ end using poly(A) polymerase (New England BioLabs), copied into cDNA using M-MLV reverse transcriptase (RNase H minus, AffinityScript, Agilent) and an oligo-dT primer, amplified by PCR and fractioned by gel electrophoresis using an Agencourt AMPure XP kit (Beckman Coulter Genomics). Fragments of 200–500 bp were selected for sequencing, which was done using an Illumina HiSeq 2000 platform (single end, 50-bp read length). Reads were trimmed of 5′ adapter and poly(A) sequences and aligned against the genome of E. coli K-12 strain MG1655 (seq) (NCBI, accession number U00096.2).

Changes in the miRNA level of expression were evaluated by real-time RT-PCR. To proceed, total RNA was isolated from long-term exposed and time-matched controls = BEAS-2B cells using TRI Reagent^® (Invitrogen, Waltham, MA, USA), in triplicate. RNA quantity was measured on a Nanodrop spectrophotometer (Thermo Fisher Scientific Technologies, Walthan, MA, USA). RNase-free DNase I (Turbo DNA free™ kit, TermoFisher Scientific) was used to remove DNA contamination. An amount of 80 ng of total RNA in a final volume of 10 µL was used for cDNA synthesis. These 10 µL included 1 µL of 10X poly(A) polymerase buffer, 10 mM of ATP, 1 µM of RT-primer (Sigma-Aldrich, Steinheim, Germany), 0.1 mM of each deoxynucleotide (dATP, dCTP, dGTP, and dTTP) (VWR International, Ballicoolin, Dublin, Ireland), 100 units of MulV reverse transcriptase (New England Biolabs, Ipswich, MA, USA), and 1 unit of poly(A) polymerase (New England, Biolabs, Ipswich, MA, USA). The mix was incubated at 37 °C for 1 h, followed by enzyme inactivation at 95 °C for 5 min. The sequence of the RT-primer was 5′-CAGGTCCAGTTTTTTTTTTTTTTTVN, where V is A, C, and G and N is A, C, G, and T.

The 5′ terminal sequence for GLRaV-4 strain 4 and strain 5 was determined using a commercially available rapid amplification of cDNA ends [RACE] system (Version 2.0, ThermoFisher Scientific, Grand Island, NY), as described in Donda et al. [25 (link)]. For additional confirmation of the 5′ terminal nucleotide, dA-tailing method was used as described earlier by Donda et al. [25 (link)]. The 5′ terminal sequence for GLRaV-4 strain 9 was determined using FirstChoice® RLM-RACE Kit (Ambion, Austin, TX, USA), according to the manufacturer’s instructions, since the 5′ RACE system Version 2.0 mentioned above was not successful. To determine the 3′ terminal sequence of GLRaV-4 strains, A-tailing of the 3′ end of viral RNA using Poly(A) polymerase (New England Biolab, Ipswich, MA) was employed as described earlier [26 (link)]. Subsequently, C-tailing of the 3′ end of viral RNA was used employing Poly(U) polymerase (New England Biolab, Ipswich, MA) for resolving ambiguity that may occur because of the presence of “A” as the 3′-terminal nucleotide. A list of primers used in these methods is provided in Additional file Table S1.