Massively Parallel Profiling of Single-Cell RNA Dynamics Using Well-TEMP-seq.

Single-cell RNA sequencing (scRNA-seq) reveals the transcriptional heterogeneity of cells, revolutionizing our understanding of cellular processes. However, the static snapshots obtained from scRNA-seq fail to reveal the time-resolved dynamics of transcription, which impedes critical insights into various biological processes, such as cellular differentiation, embryonic development, disease progression, and responses to external stimuli. Here, we describe Well-TEMP-seq, a protocol for massively parallel profiling of the temporal dynamics of single-cell gene expression. Well-TEMP-seq combines metabolic RNA labeling with a microwell-based scRNA-seq method, Well-paired-seq, to distinguish newly transcribed RNAs marked by T-to-C substitutions from pre-existing RNAs in each of thousands of single cells. Well-TEMP-seq is high-throughput, cost-effective, accurate, and provides a low cell loss rate and high single cell/bead pairing efficiency. More importantly, Well-TEMP-seq can be easily set up in other labs, and the loading of cells and beads can be easily accomplished by an optical microscope and a pipette. We believe that Well-TEMP-seq will be widely adopted and help researchers perform transformative research to unveil the dynamics of single-cell gene expression in diverse biological processes. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Well-paired-seq chip fabrication Basic Protocol 2: Well-TEMP-seq sample processing Basic Protocol 3: Bioinformatics analysis.