*Result*: Heterogeneous and novel transcript expression in single cells of patient-derived clear cell renal cell carcinoma organoids

*Splicing is often dysregulated in cancer, leading to alterations in the expression of canonical and alternatively spliced isoforms. We used the multiplexed arrays sequencing (MAS-seq) protocol of PacBio to sequence full-length transcripts in patient-derived organoid (PDO) cells of clear cell renal cell carcinoma (ccRCC). The sequencing revealed a heterogeneous dysregulation of splicing across 2599 single ccRCC cells. The majority of novel transcripts could be removed with stringent filtering criteria. The remaining 31,531 transcripts (36.6% of the 86,182 detected transcripts on average) were previously uncharacterized. In contrast to known transcripts, many of the novel transcripts have cell-specific expression. Novel transcripts common to ccRCC cells belong to genes involved in ccRCC-related pathways, such as hypoxia and oxidative phosphorylation. A novel transcript of the ccRCC-related gene nicotinamide N-methyltransferase is validated using PCR. Moreover, >50% of novel transcripts possess a predicted complete protein-coding open reading frame. An analysis of the most dominant transcript-switching events between ccRCC and non-ccRCC cells shows many switching events that are cell- and sample-specific, underscoring the heterogeneity of alternative splicing events in ccRCC. Overall, our study elucidates the intricate transcriptomic architecture of ccRCC, underlying its aggressive phenotype and providing insights into its molecular complexity.*