Ribogreen assay

DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen) and quantified with the PicoGreen assay (Thermo Fisher Scientific). RNA was extracted using the RNeasy Mini kit (Qiagen) and quantified with the RiboGreen assay (Thermo Fisher Scientific). A Bioanalyzer (Agilent Technologies) and Fragment Analyzer (Advanced Analytics) were used to characterize DNA and RNA quality and integrity. We then prepared samples for whole-exome sequencing (WES) and RNA sequencing (RNA-seq).

ON-TARGETplus Non-targeting Control Pool or ON-TARGETplus SMARTPool siRNAs targeting mouse Tlr4, Nr1h3, Nr1h2, Eif2ak3, or Ero1l were purchased from Horizon Discovery Ltd. (Cambridge, UK). All siRNAs were packaged in lipid nanoparticles (LNPs) and transfected into cells. Cultured cells were transfected with siRNA for 24 h and then treated with cholesterol or 7-KC 12 h after transfection was completed. siRNA-loaded LNPs were formulated as previously described^{53 (link)}. Briefly, 400 μL of lipid solution containing 1.875 mM ssPalmO-Phe (Product# COATSOME^® SS-OP, NOF CORPORATION, Tokyo, Japan), 450 μM polyethylene glycol-dimyristoylglycerol (Product# SUNBRIGHT^® GM-020, NOF CORPORATION), and 1.875 mM cholesterol (Sigma–Aldrich) in ethanol was prepared. Then, 10 μg of siRNA in 124 μL of 20 mM malic acid buffer (pH 3.0) was gradually added to the lipid solution under vigorous mixing. The mixed solution was diluted with 1 mL of malic acid buffer and then mixed vigorously with 3 mL of PBS. This diluted solution was subjected to ultrafiltration with Amicon Ultra-4 (Merck Millipore, Billerica, MA) two or more times. The encapsulation efficiency and recovery ratio of siRNA in this LNP solution were measured with a RiboGreen assay (Thermo Fisher Scientific Inc.) as previously described^{54 (link)}.

For cohort 1 (cases 01–12), tissue samples were snap frozen in liquid nitrogen at the time of surgery and stored at −80 °C. Tumor and matched normal (from peripheral blood or non-neoplastic tissue) DNA and tumor RNA were extracted using the DNeasy Blood and Tissue Kit (Qiagen, Venlo, the Netherlands) and the RNeasy Plus Mini Kit (Qiagen), respectively. For cohort 2 (cases 13–40), FFPE tumor tissue was sectioned and stored at room temperature. DNA and RNA were extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen) and the RNeasy FFPE kit (Qiagen), respectively. DNA was quantified with Nanodrop 8000 (Thermo Fisher Scientific, Waltham, MA, USA) and the PicoGreen assay (Thermo Fisher Scientific). RNA was quantified with the RiboGreen assay (Thermo Fisher Scientific).

The RT–qPCR was performed in a 25 μl volume containing 6.25 μl of 4X TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher Scientific), 0.5 μl of 20 μM forward and reverse primers, 0.5 μl of 20 μM probes, 5 μl of viral RNA being tested, and 12.5 μl RNAse-free water using the QuantStudioTM 7 Flex System (Applied Biosciences). The primers (D2-1929, cD2-2116) and probe (D2-2000VIC-MBG/NFQ quencher) anneal at envelope (E) gene of the DENV-2 and their sequences have been reported previously [10 (link), 14 (link)]. The viral copy number was determined using a standard curve generated by an in-vitro transcribed DENV-2 RNA as previously described [10 (link)]. The concentration of standard RNA was determined based on the Certificate of Analysis received from Pharmaceutical Product Development, a global contract research organization. Briefly, a high concentration stock of DENV-2 RNA transcript obtained from CDC was tested by droplet digital PCR and was further diluted and quantified by RT–qPCR against standards previously measured by Ribogreen assay (Thermo Fisher Scientific) to make stocks at 2.7 × 10⁷ copies/μl. The stock was 10-fold serially diluted to generate the standard curve in the RT–qPCR to measure the concentration of the samples.