Long-Read Sequencing Reveals Cell- and State-Specific Alternative Splicing in 293T and A549 Cell Transcriptomes

Alternative splicing (AS) is a fundamental mechanism governing transcriptomic diversity and cellular identity. Although 293T (human embryonic kidney) and A549 (human lung adenocarcinoma) cell lines are widely used, cell-type-specific splicing dynamics-including responses to receptor overexpression-remain incompletely characterized. To address this, we integrated Oxford Nanopore long-read sequencing with BGI short-read data to profile transcriptomes under both basal and GPCR-overexpressing conditions (<i>ADORA</i>3 in 293T; <i>P2RY12</i> in A549). Full-length isoform analysis using FLAIR and SQANTI3 revealed extensive transcriptomic complexity, including 18.02% novel isoforms in 293T and 19.52% in A549 cells. We found that 293T cells exhibited a stable transcriptome architecture enriched in splicing-related pathways, whereas A549 cells underwent broader transcriptional remodeling linked to tumorigenic processes. These findings suggest that 293T cells may be a suitable model for investigating splicing regulation, while A549 cells could serve as a relevant system for exploring tumor-related transcriptome dynamics. Our work elucidates context-dependent AS regulation and underscores the value of integrating long-read sequencing with FLAIR/SQANTI3 for dissecting cell-state-specific transcriptome dynamics.