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

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.