Fluorouridine labeling enables spatiotemporal mapping of transcriptomes and enhancers in vivo.

Transient RNA-labeling tools can reveal transcriptional dynamics with high temporal precision, yet most approaches remain largely confined to cell culture. Here, we report a 5-fluorouridine (5-FUd)-based transient transcriptomics method, 5FU-seq, that surpasses standard strategies by enabling higher metabolic incorporation, greater sensitivity, and robust detection across a broad spectrum of transcripts. Applying 5FU-seq to the murine brain uncovered stimulus-dependent transcriptional networks and revealed a dynamic regulatory program associated with Nrf2 regulation in the hippocampus during anesthesia-induced unconsciousness. We further engineered a transgenic mouse line for cell type-specific transient transcriptomics in situ, enabling simultaneous mapping of gene expression and active enhancer activity with high spatiotemporal resolution. Benchmarking in myeloid cells responding to inflammatory cues against FACS-based RNA-seq and ATAC-seq confirmed the robustness of this dual chemical-genetic toolkit. Together, this work establishes a broadly applicable platform for integrated transcriptomic and epigenetic profiling in complex tissues.