Optimizing Single-Cell Long-Read Sequencing for Enhanced Isoform Detection in Pancreatic Islets [sc_islet]

The goal of this study was to optimize single-cell long-read RNA sequencing in pancreatic islets to enable accurate isoform-level gene expression analysis. We aimed to overcome key technical limitationsincluding insufficient read lengths, high error rates, and transcript abundance biasthat have hindered long-read sequencing in single cells. Specifically, we compared 5 and 3 library preparation strategies and evaluated transcript depletion methods to improve detection of low-abundance isoforms. Our objective was to establish a robust protocol that enhances isoform identification and reveals differential transcript usage across pancreatic islet cell types. Single-cell RNA-seq libraries from dissociated pancreatic islets of 10-week-old C57BL/6 mice. We generated three libraries: one generated with 3 10x Genomics technology, one generated with 5 10x Genomics technology, and one generated with 5 10x Genomics technology with protocol modifications (extention time and dNTP concentration). The 3' library was sequenced on a Illumina short-read sequencer. The two 5' libraries were sequenced on an Oxford Nanopore long-read sequencer.