A convenient single-cell assay for the newly synthesized transcriptome reveals FLI1 regulon downregulation during T cell activation.

Understanding transcription dynamics in rapidly changing systems requires separating information about newly synthesized transcripts from bulk transcript data. Here, we developed newly synthesized transcriptome on 10× expression sequencing (NOTE-seq), a method for simultaneous profiling of regular and newly synthesized transcriptomes in single cells with high cellular throughput. NOTE-seq integrates 4-thiouridine labeling of newly synthesized RNA, thiol-alkylation-based chemical conversion, and a streamlined 10× Genomics workflow, making it accessible and convenient for biologists without extensive single-cell expertise. Using NOTE-seq, we investigated the temporal dynamics of gene expression during early-stage T cell activation, identified transcription factors and regulons in Jurkat and naive T cells, and revealed that FLI1 downregulation is a key event during T cell stimulation. Notably, topoisomerase inhibition led to the depletion of both topoisomerases and FLI1 in T cells through a proteasome-dependent mechanism. This degradation was driven by topoisomerase cleavage complexes rather than topoisomerase catalytic inhibition, highlighting potential complications topoisomerase-targeting cancer chemotherapies could pose to the immune system.