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. Overall design: eHAP cells were cultivated and subjected to NOTE-seq and 10xGenomics 3' expression v3.1 kit. Jurkat T cells were cultivated and subjected to PMA/ION treatment versus DMSO treatment, followed by NOTE-seq profiling. Mouse naïve T cells were subjected to antiCD3 and antiCD28 stimulation, followed by NOTE-seq profiling. For the barnyard experiment, Jurkart T cells and NIH3T3 cells were mixed at a 1:1 ratio, followed by the NOTE-seq workflow without T-to-C conversion. newly synthesized transcriptome profiling at single cell level for eHAP1 cells fixed with formaldehyde (NOTE-seq) or methanol (MeOH-4sU-10X). NOTE-seq profiling of K562 cells with or without chemical conversion. NOTE-seq profiling of Jurkat cells with or without PMA/ION stimulation