Abstract
Transcriptome analysis in single cells, enabled by single-cell RNA sequencing, has become a prevalent approach in biomedical research, ranging from investigations of gene regulation to the characterization of tissue organization. Over the past decade, advances in single-cell RNA sequencing technology, including its underlying chemistry, have significantly enhanced its performance, marking notable improvements in methodology. A recent development in the field, which integrates RNA metabolic labeling with single-cell RNA sequencing, has enabled the profiling of temporal transcriptomes in individual cells, offering new insights into dynamic biological processes involving RNA kinetics and cell fate determination. In this review, we explore the chemical principles and design improvements that have enhanced single-molecule capture efficiency, improved RNA quantification accuracy, and increased cellular throughput in single-cell transcriptome analysis. We also illustrate the concept of RNA metabolic labeling for detecting newly synthesized transcripts and summarize recent advancements that enable single-cell temporal transcriptome analysis. Additionally, we examine data analysis strategies for the precise quantification of newly synthesized transcripts and highlight key applications of transcriptome and temporal transcriptome analyses in single cells.