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bioRxiv · 10.1101/2024.08.22.609222

A convenient single-cell newly synthesized transcriptome assay reveals gene expression dynamics during early-stage T-cell activation

Lyu, J.·Xu, X.·Chen, C.
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Abstract

Sequencing newly synthesized transcriptome alongside regular transcriptome in single cells enables the study of gene expression temporal dynamics during rapid chromatin and gene regulation processes. Existing assays for profiling single-cell newly synthesized transcriptome often require specialized technical expertise to achieve high cellular throughput, limiting their accessibility. Here, we developed 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 naive 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 driven by topoisomerase cleavage complexes, highlighting potential complications topoisomerase-targeting cancer chemotherapies could pose to the immune system.

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Lyu, J., Xu, X., Chen, C.. 2024-08-23. A convenient single-cell newly synthesized transcriptome assay reveals gene expression dynamics during early-stage T-cell activation. https://doi.org/10.1101/2024.08.22.609222

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