Single-cell nascent transcription reveals sparse genome usage and plasticity [EU_seq]

?Understanding cell diversification from a common genome in metazoans requires single-cell transcriptional analysis. We introduce scFLUENT-seq, a single-cell nascent RNA sequencing method using brief 10-minute metabolic labeling to capture genome-wide transcription. Surprisingly, individual cells?from splenic lymphocytes to pluripotent stem cells?transcribe only 0.02~3.1% of the genome, versus >80% in bulk, revealing limited genome engagement and profound cell-type and cell-to-cell heterogeneity. Intergenic transcription, especially from heterochromatin, is pervasive and stochastic. Promoter-associated antisense and genic transcription rarely co-occur in the same cell. Proximal intergenic transcription involves both gene readthrough and independent initiation, while distal intergenic transcription is largely independent of neighboring genes and correlates with increased transcriptional diversity, a hallmark of cellular plasticity. Although global RNA sysnthesis and turnover are coupled in bulk, individual mRNA transcription and decay are poorly coordinated in single cells, suggesting noise-buffering mechanisms. Overall, scFLUENT-seq uncovers complex coding and noncoding transcriptional dynamics that underlie single-cell heterogeneity and state transitions. Overall design: For 5-ethynyl uridine (EU) labeling, mouse embryonic stem cells (ESCs) were cultured for 10 min with the medium containing 2 mM EU, washed with ice-cold PBS, and then collected by centrifugation after ice-cold trypsin digestion. 106 fixed nuclei of mouse ESCs were prepared for in-situ click and reverse transcription, followed by biotinylated RNA pull-down and RNA library contruction.