5 race

Overnight cultures of V. cholerae were back-diluted 1:100 and incubated at 37°C for 3 h before lysing. Three independent cultures of T6-active V. cholerae C6706 qstR* and 3223-74 WT were harvested by centrifugation at room temperature. RNA isolation, genomic DNA removal, and RNA cleanup were performed as previously described (59 (link)). Genomic DNA contamination was confirmed by conducting PCR with primer pair specific for 16S rRNA loci (rrsA) as previously described (Table S3) (60 (link)). RNA purity was confirmed by NanoDrop (260/280 ≈ 2.0).
5′-RACE (Invitrogen, MA, USA) was conducted according to the manufacturer’s protocol with slight modifications. Specifically, SuperScript IV reverse transcriptase (Invitrogen, MA, USA) was used to complete the first strand cDNA synthesis. Two vipA-specific primers (GT3056 and GT3060) were used to identify the +1 of transcription for the major T6 gene cluster (Table S3). PCR products were purified with QIAquick PCR purification kit (Qiagen, Hilden, Germany) or Zymoclean gel DNA recovery kit (Zymo Research, CA, USA). Sanger sequencing was conducted by Eton Bioscience Inc. (NC, USA) with the corresponding nesting primer (Table S3).

A DNA BLAST search of NCBI database was conducted using BbtTLR1 sequence from B. belcheri (GenBank: DQ400125.2). We obtained a sequence (GenBank: AF391294.1) from B. floridae showing 82% identity. In addition, a DNA BLAST search using bbtTLR1 was performed in the genome scaffold of B. lanceolatum and we identified a short sequence (ContigAmph29716) showing 83% identity. The forward primer (Table 1) was designed based on the conserved region between bbtTLR1 B. belcheri and B. floridae sequence. The reverse primer (Table 1) was designed based on the ContigAmph29716 sequence. We cloned a fragment of around 2,000 bp by PCR using the cDNA prepared from the whole animal. The 5′-end was obtained by 5′ RACE (Invitrogen) using gene specific primers (Table 1). A fragment of ~600 bp was obtained. The 3′-end was obtained by 3′ RACE (Invitrogen) using gene specific primer (Table 1). A fragment of ~1,000 bp was obtained. Finally, a PCR amplification was carried out to obtain the full-length sequence with Expand high fidelity PCR system (Roche) using the full-length primers (Table 1) designed in the non-coding regions from both 5′ to 3′-ends. All the fragments were separated by electrophoresis and cloned into the pGEM-T Easy Vector (Promega). Sequencing was carried out using T7 and SP6 primers (Servei de Genòmica i Bioinformàtica, IBB-UAB).

RNA was extracted from rescued viruses, cDNA was generated and sequencing [44] (link) of the complete genomes of all viruses was performed to confirm the genetic identity of all viruses utilized for avian virulence testing. Sequencing of WNV cDNA within plasmids, and overlapping cDNA fragments amplified from viral RNA genomes by RT-PCR, was performed using previously described protocols [45] (link). The extreme 5′-terminal sequence of each WN/IC NS3-249 mutant RNA genome was determined by using the 5′RACE (Invitrogen) method [42] . Similarly, the extreme 3′-terminal sequence was determined by employing E. coli poly(A) polymerase to tail the RNA with poly(A), followed by RT-PCR using virus-specific and oligo(dT) primers, as described previously [46] (link). Total RNA was extracted directly from AMCR sera at 4 or 5 dpi using Trizol LS (Invitrogen) or a viral RNA extraction kit (Qiagen) to avoid confounding cell culture passage-related sequence changes. RNA was reverse transcribed and PCR amplified to generate a 900 nt amplicon using a 1-step RT-PCR kit (Invitrogen). The primers used to reverse transcribe the cDNA template and DNA amplicons were: forward primer- 5′-CAGGGTGAAAGGATGGATGAG-3′ and reverse primer- 5′-CACCAACTTGCGACGGATTTG-3′. All WNV amplicons were sequenced using a capillary sequencer.

Plasmid containing cDNAs encoding X. laevis vgll2 (IMAGE clone 4930090, accession number BC056001) and ets1 (IMAGE clone 8549297, NM_001087613) were obtained from Geneservice and Source BioScience, respectively. cDNA encoding Xenopus laevis vgll3 (XL405a05ex, accession number BP689606) was obtained from the National BioResource Project (www.nbrp.jp). The 5′-sequence of vgll3 mRNA was obtained by 5′-RACE (Invitrogen). Coding sequences for tead1, tead2 and ets1 were subcloned in pCS2+MT vector. Cloning strategies for HA-vgll3 cDNAs are indicated in Table S1.

The transcription start site of sslE was determined using 5′ RACE (Version 2.0; Invitrogen) [59 (link)]. Exponentially growing cells (OD_600nm = 0.6) were stabilized with two-volumes of RNAprotect Bacteria Reagent (Qiagen) prior to RNA extraction using the RNeasy Mini Kit (Qiagen) with optional on-column DNase digestion. First-strand cDNA was synthesized and PCR amplified using the following gene specific primers: 4207, 4208 and 4209 following manufacturer’s specification. Amplified cDNA ends were sequenced to determine the transcription start site.

Determination of the transcriptional start point of mptA was performed by 5’RACE® (Invitrogen, Life Technologies, Darmstadt, Germany), using cDNA synthesized from RNA of TPEN treated cultures with gene specific primer ocDNAmptA. Briefly, RNA was treated with Terminator 5’-Phosphate-Dependent Exonuclease (TEX, Epicentre, Madison WI, USA) prior cDNA synthesis, to digest degraded mRNA transcripts. Following, an oligo-dC tail was attached by using terminal deoxynucleotidyl transferase (TdT, Invitrogen, Life Technologies, Darmstadt, Germany). The tailed cDNA was amplified by use of a nested gene specific primer (oGSP1mptA) and 5’ RACE® Abridged Anchor primer (AAP). PCR products were cloned into pJet™1.2 (ThermoFisher Scientific, Waltham, MA, USA) and plasmids of three transformants were submitted to sequencing (Seqlab, Göttingen, Germany).