Simultaneous Measurement of RNA Synthesis and Degradation Rates in Single Cells Unveils the Regulatory Mechanisms of Temporal RNA Dynamics

Abstract The temporal dynamics of coordinated RNA transcription and degradation are heterogeneous among different cell types or states. However, current transcriptomic approaches are unable to simultaneously measure transcriptome‐wide RNA synthesis and degradation rates in single cells for resolving the RNA regulatory mechanisms during dynamic processes. Herein, we develop scDUAL‐seq, which integrates double nucleoside analogs pulse‐pulse labeling, chemical nucleoside conversion, and single‐cell RNA sequencing to record both RNA transcription and RNA degradation in heterogeneous cell populations. Compared to single nucleoside analog labeling‐based methods, scDUAL‐seq allows simultaneous recording of RNA transcription and degradation, leading to more accurate determination of RNA kinetic rates and RNA regulatory strategies. In a cortisol response model, scDUAL‐seq enables the discovery of key regulons driving strategy transitions and elucidation of the coordinated regulation of RNA synthesis and degradation rates that control distinct strategy transitions. scDUAL‐seq is expected to provide new insights into RNA regulatory mechanisms across diverse biological processes.