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bioRxiv · 10.1101/2025.06.03.657745

Highly sensitive and scalable time-resolved RNA sequencing in single cells with scNT-seq2

Qiu, Q.·Zhang, H.·Gao, W.·Li, F.·Liang, D.·Wu, H.
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Abstract

Understanding gene expression dynamics requires resolving newly synthesized RNAs from pre-existing pools at single-cell resolution. Here, we present scNT-seq2, a highly sensitive and scalable method for time-resolved single-cell RNA sequencing. By systematically optimizing the second-strand cDNA synthesis (2nd SS) step, we substantially improved read alignment rates, reduced background mutations, and enhanced library complexity compared to the original scNT-seq 1. Benchmarking in 4sU-labeled K562 cells demonstrated that scNT-seq2 accurately quantifies newly synthesized transcripts and preserves the gene level RNA turnover. The enhanced sensitivity enables robust detection of dynamic, cell-cycle state specific genes, such as S-phase regulators. Together, scNT-seq2 provides an efficient and versatile tool for dissecting transcriptional dynamics across diverse biological systems at single-cell resolution.

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Qiu, Q., Zhang, H., Gao, W., Li, F., Liang, D., Wu, H.. 2025-06-03. Highly sensitive and scalable time-resolved RNA sequencing in single cells with scNT-seq2. https://doi.org/10.1101/2025.06.03.657745

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