Protocol to track single-cell-derived clones using DNA barcoding combined with single-cell RNA sequencing

Hee Jin Shin; Sahil Sharma; Sarah Kronheim; May Cheung; Long V Nguyen

doi:10.1016/j.xpro.2025.104229

Protocol to track single-cell-derived clones using DNA barcoding combined with single-cell RNA sequencing

STAR Protoc. 2025 Nov 27;6(4):104229. doi: 10.1016/j.xpro.2025.104229. Online ahead of print.

Authors

Hee Jin Shin¹, Sahil Sharma², Sarah Kronheim², May Cheung², Long V Nguyen³

Affiliations

¹ Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, Canada.
² Division of Medical Oncology and Hematology, Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 2M9, Canada.
³ Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, Canada; Division of Medical Oncology and Hematology, Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 2M9, Canada; Department of Medicine, University of Toronto, Toronto, ON M5S 3H2, Canada. Electronic address: long.nguyen@uhn.ca.

Abstract

Clonal fitness and plasticity drive tumor heterogeneity contributing to disease progression and treatment resistance. Here, we provide a protocol to track the clonal outputs from uniquely marked cells. We describe steps for constructing and validating lentiviral DNA barcode libraries. We then detail procedures for single-cell RNA sequencing. This approach links clonal growth activity with transcriptomic profiles and enables permanent cellular labeling to track clonal dynamics in various model systems, which can provide insight into molecular processes underlying clone function. For complete details on the use and execution of this protocol, please refer to Nguyen et al.¹.

Keywords: Cancer; Cell Biology; RNA-seq; Sequence analysis; Sequencing; Single Cell.