GoT-Splice protocol for multi-omics profiling of gene expression, cell-surface proteins, mutational status, and RNA splicing in human cells

• Published in: STAR protocols 2024-06, Vol.5 (2), p.102966, Article 102966
• Main Authors: Ganesan, Saravanan, Cortés-López, Mariela, Swett, Ariel D., Dai, Xiaoguang, Hickey, Scott, Chamely, Paulina, Hawkins, Allegra G., Juul, Sissel, Landau, Dan A., Gaiti, Federico
• Format: Article
• Language: English
• Subjects: Gene Expression; Gene Expression Profiling - methods; Gene Library; Genomics; Humans; Membrane Proteins - genetics; Membrane Proteins - metabolism; Molecular Biology; Multiomics; Mutation - genetics; Proteomics - methods; Protocol; RNA Splicing - genetics; RNA-seq; Sequencing; Single Cell; Single-Cell Analysis - methods; Transcriptome - genetics
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2024.102966

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 [Display omitted] •GoT-Splice profiles single-cell genotype, expression, surface markers, and splicing•ONT-sc-Splice quantifies splicing junctions for comparative cell analysis•Re-amplification of 10× library cDNA, followed by sequential PCRs•Pull-down of the target locus and indexing of the sample for sequencing Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.