biostudies-literatureUnknown5(2)Ganesan SStudying RNA splicing factor mutations is challenging due to difficulties in distinguishing wild-type and mutant cells within complex human tissues and inaccuracies associated with reconstructing splicing signals from short-read sequencing data. Here, we present Genotyping of Transcriptomes (GoT)-Splice, a protocol that overcomes these limitations by combining GoT with enhanced long-read single-cell transcriptome and cell-surface proteomics profiling. We describe steps for long-read library preparation and analysis, followed by cDNA re-amplification, enrichment of mutation of interest, sample indexing, and GoT library preparation. For complete details on the use and execution of this protocol, please refer to Cortés-López et al.¹.STAR protocols102966https://www.ebi.ac.uk/biostudies/studies/S-EPMC10966806biostudies-literatureGoT-Splice protocol for multi-omics profiling of gene expression, cell-surface proteins, mutational status, and RNA splicing in human cells.PMC10966806Chamely PGaiti FHickey SGanesan SHawkins AGSwett ADJuul SCortes-Lopez MDai XLandau DAfalseGoT-Splice protocol for multi-omics profiling of gene expression, cell-surface proteins, mutational status, and RNA splicing in human cells.Studying RNA splicing factor mutations is challenging due to difficulties in distinguishing wild-type and mutant cells within complex human tissues and inaccuracies associated with reconstructing splicing signals from short-read sequencing data. Here, we present Genotyping of Transcriptomes (GoT)-Splice, a protocol that overcomes these limitations by combining GoT with enhanced long-read single-cell transcriptome and cell-surface proteomics profiling. We describe steps for long-read library preparation and analysis, followed by cDNA re-amplification, enrichment of mutation of interest, sample indexing, and GoT library preparation. For complete details on the use and execution of this protocol, please refer to Cortés-López et al.¹.2024-01-01T00:00:00Z2024 Mar2025-04-21T21:28:34.169Z2025-04-05T18:25:05.145ZS-EPMC109668063851286710.1016/j.xpro.2024.102966