Sequencing of newly synthesized RNA can monitor transcriptional dynamics with great sensitivity and high temporal resolution, but is currently restricted to populations of cells. Here, we developed newly synthesized alkylation-dependent single-cell RNA sequencing (NASC-seq), to monitor both newly synthesized and pre-existing RNA in single cells. We validated the method on pre-alkylated exogenous spike-in RNA, and by demonstrating that more newly synthesized RNA was detected for genes with known high mRNA turnover. Importantly, NASC-seq reveals rapidly up- and down-regulated genes during the T-cell activation, and RNA sequenced for induced genes were essentially only newly synthesized. Moreover, the newly synthesized and pre-existing transcriptomes after T-cell activation were distinct confirming that we indeed could simultaneously measure gene expression corresponding to two time points in single cells. Altogether, NASC-seq is a powerful tool to investigate transcriptional dynamics and it will enable the precise monitoring of RNA synthesis at flexible time periods during homeostasis, perturbation responses and cellular differentiation.