HepG2 RNA-seq

For the transcriptomic profiling of the HepG2, LX2, and RAW 264.7 cell lines, total RNA was extracted using the RNeasy Mini Kit (Qiagen) following the manufacturer’s protocols. RNA concentration and purity were determined via NanoDrop spectrophotometry, and RNA integrity (RIN) was confirmed using an Agilent 2100 Bioanalyzer to ensure suitability for high-depth sequencing. Purified RNA samples from the in vitro experiments were submitted to the Auburn University Center for Comparative Medicine (AUCCM) Genomics & Sequencing Core for library preparation and analysis. Sequencing libraries were constructed using the Illumina Stranded mRNA Prep kit to specifically target the protein-coding transcriptome. The libraries were then sequenced on an Illumina platform to generate 150-bp paired-end reads, achieving a target depth of 25–30 million reads per sample. The resulting raw FASTQ files were processed using a bioinformatics pipeline identical to that used for the in vivo tissue samples. Initial quality control was performed with FastQC, followed by adapter trimming and quality filtering. Reads were aligned to the appropriate reference genome (GRCh38 for human cell lines; mm10 for murine cells) using the STAR aligner. Gene-level counts were generated via featureCounts, and differential gene expression was analyzed using the DESeq2 package in R. Significance was defined by an adjusted p-value (FDR) 1.0.