Ambion megascript t7 kit

We prepared dsRNA for RNAi experiments as previously described [30 (link)]. Briefly, the A. pisum p300/CBP mRNA sequence was used as a template and gene-specific RNAi primers including a 5′ T7 promoter were designed using Primer3 v4.1.0 and were purchased from Sigma-Aldrich (Germany). The dsRNA construct was designed to be 367 bp in length (GC content = 40%–60%) covering part of the ORF (Figure 1, Table S1). The construct was checked for off-targets by screening against the entire pea aphid genome, ensuring there were no overlaps >19 bp with other A. pisum genes. The PCR amplicon generated using the RNAi primers and cDNA template was cloned and sequenced as described above. The verified plasmid vector was used as a PCR template for the RNAi primers and the amplicon was excised from the gel and purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey–Nagel). The purified PCR product was used to synthesize dsRNA with the Ambion MEGAscript T7 kit (Applied Biosystems, USA). The dsRNA was purified by isopropanol precipitation and washed with ethanol. The pellet was resuspended in 30–50 µL nuclease-free water and stored −20 °C. Primers and accession numbers for all p300/CBP sequences used in this study are listed in Table S1.

Double stranded RNA (dsRNA) for Col1 was prepared using the Ambion MEGAscript T7 kit (Applied Biosystems) according to the manufacturer’s protocol. A 320-bp template for dsRNA was generated by PCR with gene-specific primers (Supplementary information, Figure S1) including the T7 polymerase promoter sequence at the 5′ end (forward: 5′-TAA TAC GAC TCA CTA TAG GGA GTT GCC TGC ATC TCC TTC CAA T-3′, and reverse: 5′-TAA TAC GAC TCA CTA TAG GGA GTT AGC TTT GCC TGG TCC TCT G-3′). Adult H. axyridis beetles from the invasive population sourced in Germany were injected with 1 µg of Col1 dsRNA (250 nl total volume). Controls were injected with the same volume of water. After 2 days, beetles were infected with 4 µl of a mixture of live bacteria containing 1.7 × 10⁹ cfu/ml E. coli and 1.1 × 10⁹ cfu/ml M. luteus. The experiment was carried out four times and five immune-challenged Col1-RNAi specimens, water controls and untreated controls per replicate were pooled and analysed by qPCR at 24 h post-infection (hpi).