BioProject

Single-cell nascent RNA sequencing is essential for understanding how a genome drives cell diversity. More...

Single-cell nascent RNA sequencing is essential for understanding how a genome drives cell diversity. We developed scFLUENT-seq, a single-cell method that captures genome-wide transcription with brief metabolic labeling. Our analysis shows that only 3~6% of the genome is transcribed per cell in a 10-minute window, compared to over 80% in bulk, revealing significant variability in how individual cells interpret the genome. Notably, substantial transcription occurs in intergenic regions, particularly in heterochromatin, with high stochasticity. Moreover, promoter-associated antisense and genic sense transcription rarely co-occur in the same cell. Distal intergenic transcription correlates poorly with gene activity but links to increased genome-wide transcriptional diversity, which marks cellular plasticity and may precede cell-state shifts. Furthermore, mRNA synthesis and decay are uncoupled at the single-cell level, unlike intergenic ncRNA, suggesting specialized mechanisms counteracting stochastic noncoding production. In summary, scFLUENT-seq captures the full transcriptional spectrum, revealing the heterogeneity and regulatory complexity underlying cellular plasticity. 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. Less...