GSE128273
NASC-seq monitors RNA synthesis in single cells
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 develop new transcriptome alkylation-dependent single-cell RNA sequencing (NASC-seq), to monitor newly synthesized and pre-existing RNA in single cells. We validate the method on pre-alkylated RNA, and by demonstrating that more newly synthesized RNA was detected for genes with known high mRNA turnover. NASC-seq reveals rapidly up- and down-regulated genes during T-cell activation, and RNA arising from induced genes is essentially only newly synthesized. The newly synthesized and pre-existing transcriptomes after T-cell activation are distinct, confirming that we simultaneously measure gene expression at two time points in single cells. Altogether, NASC-seq is an accessible and powerful tool to investigate transcriptional dynamics that enables the precise monitoring of RNA synthesis at flexible time periods during homeostasis, perturbation responses and cellular differentiation. Overall design: For each single-cell RNA-seq experiment, 188 single cells were sequenced according to the NASC-seq protocol. The following experiments are included:  exp1: K562 cells were labelled for 60 minutes with 50uM 4sU. Negative controls and unlabelled cells are included in the experiment  exp2: Jurkat cells were stimulated with PMA and ionomycin and simultaneously labelled with 50uM 4sU for 15 minutes. Negative controls, unlabelled cells and unstimulated cells are included in the experiment.  exp3: Jurkat cells were stimulated with PMA and ionomycin and simultaneously labelled with 50uM 4sU for 30 minutes. Negative controls, unlabelled cells and unstimulated cells are included in the experiment.  exp4: Jurkat cells were stimulated with PMA and ionomycin and simultaneously labelled with 50uM 4sU for 60 minutes. Negative controls, unlabelled cells and unstimulated cells are included in the experiment.  TT-seq: Jurkat cells were stimulated with PMA and ionomycin for 15 or 30 minutes and labelled with 500uM 4sU during the last 5 minutes of stimulation. Biological duplicates of TT-seq samples were sequenced.