5 race kit

Primer extension analysis was performed using the 5’ RACE kit (Invitrogen) according to the manufacturer’s instructions. Briefly, cDNA synthesis was performed using gene specific primers (GSP) listed in Table 1. cDNA was purified and used in TdT reactions that added a poly-cytosine tail to the 5’ end of the cDNA. Tailed cDNA was subsequently used in PCR reactions with an internal GSP primer (ackA-GSP2 or pta-GSP2—Table 1) and an abridged anchor primer provided by Invitrogen. PCR products were analyzed on an agarose gel, purified on a Qiaquick PCR purification column (Qiagen) and subjected to Sanger sequencing (ACGT Inc.).

The 5′ RACE analysis was performed according to the 5′ RACE Kit (Invitrogen) protocol and using the reagents from the kit. Total RNA was extracted from adult M. martensii cephalothoraxes using TRIzol Reagent (Life Technologies, Carlsbad, CA, USA). The RNA was subjected to reverse transcription using SuperScript II at 42 °C for 50 min, and incubated at 70 °C for 15 min to terminate the reaction. RT–PCR was carried out under the following cycling conditions: an initial denaturation of 2 min at 94 °C followed by 30–35 cycles of denaturation at 94 °C for 30 s, annealing at 55–60 °C for 30 s and extension at 72 °C for 30 s, with a final extension at 72 °C for 10 min.

Overlapping RT-PCRs were done to recover the complete genome. All primer sequences can be found in the S3 Table. The NS5, envelope and NS5-partial 3’UTR regions were first amplified using flavivirus consensus or West Nile specific primers [1 (link),105 (link),106 (link)], followed by amplification of NS3 region using designed WNV primers. The 5’ non-coding region of the genome was obtained using the 5’RACE kit (Invitrogen, Carlsbad, USA) and a designed consensus primer in the capsid protein for reverse primer. Finally, specific primers were designed according to the first sequences obtained and a second step of RT-PCR was done to obtain the complete genome.
The PCR fragments were obtained using AMV reverse transcription kit (Promega, Madison, USA) for reverse transcription and Go-Taq PCR kit (Promega, Madison, USA) for amplification. The RT conditions were set according to the manufacturer’s instructions, and the PCR conditions were as follows: 5 minutes at 95°C, 40 cycles of 1 minute at 95°C, 1 minute at 53°C, 1 to 4 minutes (according the size of the PCR product) at 72°C, and 10 minutes at 72°C. The PCR products were purified from the agarose gel using the Gel extraction kit (Qiagen) and sequenced by Cogenics (Beckman Coulter Genomics, Essex, UK).

To determine the transcription initiation site (TIS) of the gumB gene, the 5′-RACE method was carried out with the gumB sequence-specific primer gumRTP1-3 (Supplementary Table 1). The assay was performed as previously described15 (link). Briefly, total cellular RNA was extracted from the Xcc strains grown in NY medium to an OD₆₀₀ of 1.0. cDNA fragments were obtained using the 5′-RACE kit (Invitrogen Life Technologies, San Diego, CA, USA), and the PCR products were cloned into the pMD19-T vector and sequenced.

Transcription start points were identified by using a 5′ RACE kit (Invitrogen) in accordance with the manufacturer’s instructions (version 2.0) and as described previously (61 (link)), using 5 μg of total RNA for first-strand cDNA synthesis and the gene-specific primers listed in Table 1.