Project description:Understanding how the genome orchestrates cell diversity requires transcriptional analysis at single-cell resolution. Here, we introduce scFLUENT-seq, a single-cell nascent RNA sequencing technique that detects genome-wide transcription following brief metabolic labeling. Within a 10-minute window, cells from splenic lymphocytes to pluripotent stem cells engage only 0.4~10% of the genome in transcription, compared to >80% in bulk, revealing profound cell-to-cell heterogeneity. Intergenic transcription, especially from heterochromatin, is highly stochastic and pervasive. Promoter-associated antisense and genic transcription rarely co-occur in the same cell. Proximal intergenic transcription reflects both gene readthrough and independent initiation, while distal intergenic transcription is largely decoupled from neighboring gene activity and correlate with greater transcriptional diversity, a hallmark of cellular plasticity. Additionally, mRNA synthesis and decay are poorly coordinated in single cells, suggesting buffering mechanisms that constrain transcriptional noise. Overall, scFLUENT-seq captures both coding and noncoding transcriptional dynamics, revealing the regulatory complexity underlying single-cell heterogeneity and state transitions.
Project description:Understanding how the genome orchestrates cell diversity requires transcriptional analysis at single-cell resolution. Here, we introduce scFLUENT-seq, a single-cell nascent RNA sequencing technique that detects genome-wide transcription following brief metabolic labeling. Within a 10-minute window, cells from splenic lymphocytes to pluripotent stem cells engage only 0.4~10% of the genome in transcription, compared to >80% in bulk, revealing profound cell-to-cell heterogeneity. Intergenic transcription, especially from heterochromatin, is highly stochastic and pervasive. Promoter-associated antisense and genic transcription rarely co-occur in the same cell. Proximal intergenic transcription reflects both gene readthrough and independent initiation, while distal intergenic transcription is largely decoupled from neighboring gene activity and correlate with greater transcriptional diversity, a hallmark of cellular plasticity. Additionally, mRNA synthesis and decay are poorly coordinated in single cells, suggesting buffering mechanisms that constrain transcriptional noise. Overall, scFLUENT-seq captures both coding and noncoding transcriptional dynamics, revealing the regulatory complexity underlying single-cell heterogeneity and state transitions.
Project description:Understanding how the genome orchestrates cell diversity requires transcriptional analysis at single-cell resolution. Here, we introduce scFLUENT-seq, a single-cell nascent RNA sequencing technique that detects genome-wide transcription following brief metabolic labeling. Within a 10-minute window, cells from splenic lymphocytes to pluripotent stem cells engage only 0.4~10% of the genome in transcription, compared to >80% in bulk, revealing profound cell-to-cell heterogeneity. Intergenic transcription, especially from heterochromatin, is highly stochastic and pervasive. Promoter-associated antisense and genic transcription rarely co-occur in the same cell. Proximal intergenic transcription reflects both gene readthrough and independent initiation, while distal intergenic transcription is largely decoupled from neighboring gene activity and correlate with greater transcriptional diversity, a hallmark of cellular plasticity. Additionally, mRNA synthesis and decay are poorly coordinated in single cells, suggesting buffering mechanisms that constrain transcriptional noise. Overall, scFLUENT-seq captures both coding and noncoding transcriptional dynamics, revealing the regulatory complexity underlying single-cell heterogeneity and state transitions.
Project description:miRNAs are key post-transcriptional regulators of gene expression. However, it is still poorly understood how miRNAs themselves are regulated, mainly due to the sparse annotation of miRNA transcription start sites (TSSs). Here, we developed a novel method for identifying active miRNA TSSs from nascent transcriptomes generated by nuclear run-on sequencing. With the least data requirement, our method demonstrated better performance than existing methods. Moreover, it provided ways not only to recognize miRNA TSSs but also to quantify primary miRNA expression in one experiment, which is very useful for revealing miRNAs directly regulated by the regulator(s) of interest.
Project description:During maturation, eukaryotic precursor RNAs undergo processing events including intron splicing, 3’-end cleavage, and polyadenylation. Here, we describe nanopore analysis of CO-transcriptional Processing (nano-COP), a method for probing the timing and patterns of RNA processing. An extension of native elongating transcript sequencing (NET-seq), which quantifies transcription genome-wide through short-read sequencing of nascent RNA 3’ ends, nano-COP uses long-read nascent RNA sequencing to observe global patterns of RNA processing. First, nascent RNA is stringently purified through a combination of 4-thiouridine metabolic labeling and cellular fractionation. In contrast to cDNA or short-read–based approaches relying on reverse transcription or amplification, the sample is sequenced directly through nanopores to reveal the native context of nascent RNA. nano-COP identifies both active transcription sites and splice isoforms of single RNA molecules during synthesis, providing insight into patterns of intron removal and the physical coupling between transcription and splicing. The nano-COP protocol yields data within 3 days.