A convenient single-cell newly synthesized transcriptome assay reveals gene expression dynamics during early-stage T-cell activation

Abstract

Sequencing newly synthesized transcriptome alongside regular transcriptome in single cells enables the study of gene expression temporal dynamics during rapid chromatin and gene regulation processes. However, existing assays to profile single-cell newly synthesized transcriptome require in-house technical expertise to achieve high cellular throughput, limiting their widespread application. Here, we developed NOTE-seq, a method that simultaneously profiles regular and newly synthesized transcriptomes in single cells. NOTE-seq integrates 4-thiouridine labeling of newly synthesized RNA, thiol-alkylation-based chemical conversion, and a streamlined workflow on the 10X Genomics platform, offering high cellular throughput that is accessible and convenient for regular biology laboratories without specialized single-cell expertise. Using NOTE-seq, we characterized the temporal dynamics of gene expression during early-stage T-cell activation in Jurkat and naive T cells, identified transcription factors and regulons, and discovered Fli-1 as a master transcription factor for gene regulation upon T-cell stimulation. Interestingly, chemotherapeutic topoisomerase inhibitor affects Fli-1 level in T cells, indicating potential complications for the immune system.