RNA-seq of  Kupffer cell stimulated with CsEVs
Total RNA (1 g) was used for library construction with the NEB Next Ultra II RNA Library Prep Kit for Illumina or NEB Next Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA). Poly(A)-tailed mRNA was enriched using Oligo(dT) magnetic beads, followed by fragmentation via divalent cation-mediated enzymatic cleavage (RNA Fragmentation Reagents, Thermo Fisher Scientific, Waltham, MA, USA). Fragmented mRNA served as the template for first-strand cDNA synthesis using random hexamer primers. After double-stranded cDNA synthesis, purification was performed using the provided purification beads. The cDNA underwent end repair, 3'-end adenylation, and adapter ligation. Libraries were size-selected (400500 bp) using AMPure XP beads (Beckman Coulter, Brea, CA, USA), PCR-amplified, and further purified with AMPure XP beads. Library quality was evaluated using an Agilent 2100 Bioanalyzer with the Agilent High Sensitivity DNA Kit (Agilent Technologies, Santa Clara, CA, USA).