Quant it ribogreen rna reagent

DOPE, cholesterol, and DMG‐PEG2000 were purchased from Avanti Polar Lipids Inc. (Alabaster, AL). Eagle's minimum essential medium (EMEM) and other cell culture supplies were purchased from Corning Incorporated (Corning, NY). Quant‐iT RiboGreen RNA reagent and Gibco heat‐inactivated fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Waltham, MA). All the other chemical reagents were obtained from Sigma‐Aldrich or Abcam and used without further purification.

RNA was isolated from excised tumors after generation of single‐cell suspensions using an RNA isolation kit (Bio & Sell, Germany) according to the supplier's protocol. The RNA concentration of each sample was determined using fluorescent RNA labeling (Quant‐it RiboGreen RNA Reagent; Thermo Fisher, Germany) and a fluorometer (DS‐11 FX; DeNovix, Germany). Absolute concentrations were calculated against a standard curve. Subsequent gene expression analysis was performed using the NanoString nCounter technology, which directly evaluates RNA levels without prior complementary DNA synthesis. Briefly, 50 ng RNA per sample was hybridized for 24 h at 65 °C with reporter and capture ProbeSets of the nCounter PanCancer IO 360 Panel targeting 770 genes specifically designed for research in immuno‐oncology. Immediately after, samples were loaded into the nCounter SPRINT cartridge and processed by the NanoString nCounter system. Differential gene expression analysis was performed using the nSolver 4.0 analysis software (all NanoString Technologies, USA). Pathway enrichment analysis was evaluated using GSEA Software^[89, ^90] and processed with Cytoscape.^[91] Cell fractions were quantified from bulk tissue gene expression profiles using CIBERSORTx.^[92]

Liver RNA was purified and subjected to quantitative PCR analysis (supplementary material)^{50 (link)}.
Psd3 mRNA was quantitated using the primer probe set Mm01351099_m1 (ThermoFisher Scientific). RNA transcript levels were normalized to total RNA levels using Quant-iT RiboGreen RNA reagent (ThermoFisher Scientific)^{51 (link)}.
For lipogenic and inflammatory gene expression, total RNA was sequenced on a NextSeq500 sequencing instrument (Illumina) (supplementary material).

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoEthanolamine (DOPE), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K) were purchased from the NOF Corporation (Tokyo, Japan). Cholesterol was purchased from Sigma-Aldrich (St. Louis, MO, USA). A pH-sensitive cationic lipid, CL4H6, was synthesized in the laboratory as previously described.^{19 (link)} Ethanol, sodium chloride, citric acid, 2-morpholinoethanesulfonic acid (MES), monohydrate, and phosphate-buffered saline (PBS) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). The siFVII and Cy-5-labeled siGL4 were purchased from Hokkaido System Science Co. Ltd. (Sapporo, Japan). Table S4† shows the sense and antisense strand sequences of siFVII and Cy5-siGL4. Quant-iT™ Ribogreen™ RNA reagent and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) were obtained from Thermo Fisher Scientific (Waltman, MA, USA). SU-8 3050 was obtained from Nippon Kayaku Co., Ltd. (Tokyo, Japan).