5 3 race kit

For five SLRSV isolates, genome sequences, i.e. both RNA1 and RNA2, were completed from end to end (Table 1). The 3’UTR of each RNA molecule was determined by sequencing RT-PCR fragments generated using segment-specific forward primers corresponding to sequences located at the 3’ end of the ORF in combination with an oligo dT primer. The 5’UTR sequence of each RNA segment was determined using a Roche 5′/3′ RACE Kit according to the manufacturer’s protocol. In this case, the reverse primers were based on the 5’ region of the ORF of each RNA. Sanger sequencing of all amplicons that were generated was performed by Macrogen Europe (Amsterdam, The Netherlands).
The five complete annotated nucleotide (nt) sequences of SLRSV genomes and the coding sequences of additional partially sequenced isolates have been submitted to the NCBI GenBank database (see Table 1 for accession numbers).

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.

5’ RACE was performed using a 5’/3’ RACE kit (Roche, Germany) as previously described [62 (link)]. Essentially, DNase I-treated total RNA was reverse transcribed using specific primers BT3311 and BT3337 as SP1 primers for finR and fprA, respectively. The first-strand DNA (cDNA) was purified, and poly(A) was added to the 5’-terminus of the cDNA using terminal transferase. Next, poly(A)-tailed cDNA was PCR-amplified using the specific SP2 primer BT4438 for finR and BT4479 for fprA and an anchored oligo(dT) primer. The purified PCR product was cloned into the pGemT vector, and the +1 site was identified from the DNA sequences.

Bacteria used in this study (Table S1) were cultured in TY (Beringer, 1974 ) or UMS (Brown & Dilworth, 1975 ; Haskett, Paramasivan, et al., 2022b ; Poole et al., 1994 (link)). Plasmids (Table S2 and Data S2) were constructed using HiFi assembly (New England Biolabs) or BEVA modular golden‐gate assembly (Geddes, Mendoza‐Suárez, & Poole, 2019a (link); Weber et al., 2011 (link)) and were mobilized into strains of interest via di‐parental mating with E. coli ST18 (Thoma & Schobert, 2009 (link)). For mini‐Tn7 integration into the chromosome of AcLP, tri‐parental matings were required to additionally mobilize the transposase helper plasmid pTNS3 (Choi & Schweizer, 2006 (link)). 5′‐RACE was performed using a 2nd Generation Roche 5′/3′ RACE Kit as per the manufacturer's recommendations.

Total RNA was extracted from young flower buds (5–7 mm in length) with an RNeasy Plant Mini Kit (QIAGEN Sciences, Maryland, USA) in accordance with the manufacturer’s instructions. Poly(A)+ RNA was separated from total RNA using DYNABEADS (DYNAL, Oslo, Norway), and cDNA was synthesized using AMV reverse transcriptase (Roche Diagnostics GmbH, Mannheim, Germany). Partial cDNAs were isolated by 3′-rapid amplification of cDNA ends (RACE)39 using a 5′/3′-RACE Kit (Roche) and four MADS-box degenerate primers: P038, P041, AD, and SP314 (link). Upstream sequences overlapping the 3′ fragments were isolated by 5′-RACE using a 5′/3′-RACE Kit. cDNA clones with complete open reading frames (ORFs) were isolated via PCR using primers located in the 5′- and 3′-UTR regions with cDNA pools as templates. DNA sequencing was performed using a BigDye Terminator Cycle Sequencing Premix Kit (Applied Biosystems, Foster City, CA, USA) with an automated sequencer (Model 310, Applied Biosystems) according to the manufacturer’s protocol.