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

We invented 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 10X 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 naïve T cells, and uncovered the down-regulation of FLI1 as a master transcription factor upon 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.