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

Saravanan Ganesan; Mariela Cortés-López; Ariel D Swett; Xiaoguang Dai; Scott Hickey; Paulina Chamely; Allegra G Hawkins; Sissel Juul; Dan A Landau; Federico Gaiti

doi:10.1016/j.xpro.2024.102966

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

STAR Protoc. 2024 Mar 20;5(2):102966. doi: 10.1016/j.xpro.2024.102966. Online ahead of print.

Authors

Affiliations

¹ New York Genome Center, New York, NY 10013, USA; Division of Hematology and Medical Oncology, Department of Medicine and Meyer Cancer Center, Weill Cornell Medicine, New York, NY 10065, USA. Electronic address: sag4012@med.cornell.edu.
² New York Genome Center, New York, NY 10013, USA; Division of Hematology and Medical Oncology, Department of Medicine and Meyer Cancer Center, Weill Cornell Medicine, New York, NY 10065, USA.
³ Oxford Nanopore Technologies Inc., New York, NY 10013, USA.
⁴ Oxford Nanopore Technologies Inc., Alameda, CA 94501-1170, USA.
⁵ Childhood Cancer Data Lab, Alex's Lemonade Stand Foundation for Childhood Cancer, Wynnewood, PA 19096, USA.
⁶ New York Genome Center, New York, NY 10013, USA; Division of Hematology and Medical Oncology, Department of Medicine and Meyer Cancer Center, Weill Cornell Medicine, New York, NY 10065, USA; Institute for Computational Biomedicine, Weill Cornell Medicine, New York, NY 10021, USA. Electronic address: dlandau@nygenome.org.
⁷ University Health Network, Princess Margaret Cancer Centre, Toronto, ON M5G 0A3, Canada; University of Toronto, Medical Biophysics, Toronto, ON M5G 1L7, Canada. Electronic address: federico.gaiti@uhn.ca.

Abstract

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.

Keywords: Gene Expression; Genomics; Molecular Biology; RNA-seq; Sequencing; Single Cell.