Protocol for high-quality RNA sequencing, cell surface protein analysis, and genotyping in single cells using TARGET-seq+

Journal article

Studying the consequences of somatic mutations in pre-malignant and cancerous tissues is challenging due to noise in single-cell transcriptome data and difficulty in identifying the clonal identity of single cells. We optimized TARGET-seq to develop TARGET-seq+, which combines RNA sequencing (RNA-seq), the analysis of cell surface protein expression, and genotyping in single cells with improved sensitivity. We describe the steps for cell isolation, the preparation of single-cell RNA-seq (scRNA-seq) and genotyping libraries, and sequencing. We also provide guidance on the analysis of single-cell genotyping, transcriptome pre-processing, and data integration.

For complete details on the use and execution of this protocol, please refer to Jakobsen et al.¹

Authors: Jakobsen, NA; Turkalj, S; Vyas, P

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Cell Press
• Journal: STAR Protocols
• Volume: 6
• Issue: 2
• Article number: 103832
• Publication date: 2025-06-20
• Acceptance date: 2025-04-30
• DOI: 10.1016/j.xpro.2025.103832
• EISSN: 2666-1667
• Language: English
• Keywords: genomics; molecular biology; sequencing; gene expression; flow cytometry; stem cells; RNA-seq; single cell