Protocol to define Sox9 target programs during mouse limb chondrogenesis using scRNA-seq and CUT&RUN integration.

Abstract

Developing tissues comprise heterogeneous cell populations exhibiting stage-dependent transcriptional dynamics. We present a protocol to identify candidate Sox9 target genes in specific murine cell populations by integrating single-cell RNA sequencing (scRNA-seq) with Ty1-tag-based CUT&RUN. We describe steps for embryonic forelimb bud dissection, scRNA-seq and CUT&RUN sample and library preparation, and data processing. We then detail procedures for integrative analysis of stage- and cluster-specific Sox9 targets. The framework may be adaptable to other Sox9-expressing tissues and developmental stages with appropriate optimization. For complete details on the use and execution of this protocol, please refer to Sega et al.¹.