Trizol rna isolater total rna extraction reagent

First of all, C.lividipennis and A.lucorum were anesthetized using carbon dioxide. Subsequently, the SGs, guts, and other carcasses without SGs and guts from both insects were carefully dissected under a microscope (Olympus, Japan). Thereafter, the total RNA was extracted from each sample using the Trizol RNA Isolater Total RNA Extraction Reagent (Vazyme Biotech, Nanjing, China), in line with the manufacturer’s protocol. Afterwards, the RNA concentration, quantity, and integrity were assessed by the Agilent 2100 bioanalyzer (Agilent Technologies, California, USA). Then, to synthesize cDNA, the mRNA was purified from the total RNA using magnetic beads carrying Oligo(dT). The purified cDNA then experienced end repairing, adenylation of 3’ ends, and ligation of adaptors. AMPure XP beads (Beckman Coulter, Beverly, USA) were used to screen out 370–420 bp cDNA. Next, the sequencing library was generated through PCR, and AMPure XP beads were further used in a second purification step. Finally, RNA-seq was conducted on the Illumina Novaseq 6000 platform (Novogene, Tianjin, China). The quality summary regarding library sequencing is listed in (Supplementary Table 8), with over 40 million clean reads being obtained in each library.

The total RNA of the target sample was extracted using the Trizol RNA isolater Total RNA Extraction Reagent (Vazyme Biotech, Nanjing, China) following the manufacturer’s protocol. The quality and concentration of the isolated RNAs were assessed using a NanoDrop One Spectrometer (Thermo Fisher Scientific, USA). Next, 400 ng of each RNA was reverse transcripted into cDNA using the HiScript II Q RT SuperMix (+ gDNA wiper) kit (Vazyme Biotech, Nanjing, China) in line with the protocol. Finally, qRT-PCR was carried out with an ABI QuantStudio 5 equipment (Thermo Fisher Scientific, USA) in a 10-µL reaction system, containing 5µL of ChamQ SYBR Color qPCR Master Mix (Vazyme Biotech, Nanjing, China), 2.4µL of ddH₂O, 2µL of 20-fold diluted cDNA, and 0.3µL of each 10 µmol primer. The procedure of qPCR comprised an initial denaturation step at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 10 s, and primer annealing together with amplification at 60 °C for 30 s. With 2^−ΔΔCt (Ct: cycle threshold) method [58 (link)], the relative expression level of the target gene was calculated, with N. lugens 18 S ribosomal RNA (Nl18S, GenBank accession number: JN662398.1) serving as the housekeeping gene. Primers used for qRT-PCR are listed in (Supplementary Table 4 A).