An accessible and convenient assay to profile single-cell newly synthesized transcriptome reveals transcription factors and regulon dynamics during early-stage T-cell activation

Sequencing newly synthesized transcriptome in addition to the regular transcriptome in single cells enable the study of gene expression temporal dynamics during rapid chromatin and gene regulation processes. However, current single-cell newly synthesized transcriptome assays require in-house technology expertise to achieve a high cellular throughput, preventing their widespread application. Here, we develop NOTE-seq to simultaneously profile regular and newly synthesized transcriptome in single cells. NOTE-seq combines 4-thiouridine labeling of newly synthesized RNA, thiol-alkylation-based chemical conversion, and the streamlined 10X Genomics platform, offering a high cellular throughput accessible to and convenient for regular biology labs without single-cell technology expertise. Using NOTE-seq, we investigate the temporal dynamics of gene expression during early-stage T-cell activation in human Jurkat T cells and mouse naïve T cells, characterize transcription factors and regulons, and discover Fli-1 as a master transcription factor for gene regulation upon T-cell activation. Interestingly, Fli-1 level in T cells is sensitive to the treatment of camptothecin, a topoisomerase inhibitor used in cancer chemotherapy, indicating its potential complication on the immune system. 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.