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.