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NASC-seq monitors RNA synthesis in single cells.

ABSTRACT: Sequencing of newly synthesised RNA can monitor transcriptional dynamics with great sensitivity and high temporal resolution, but is currently restricted to populations of cells. Here, we develop new transcriptome alkylation-dependent single-cell RNA sequencing (NASC-seq), to monitor newly synthesised and pre-existing RNA simultaneously in single cells. We validate the method on pre-labelled RNA, and by demonstrating that more newly synthesised RNA was detected for genes with known high mRNA turnover. Monitoring RNA synthesis during Jurkat T-cell activation with NASC-seq reveals both rapidly up- and down-regulated genes, and that induced genes are almost exclusively detected as newly transcribed. Moreover, the newly synthesised and pre-existing transcriptomes after T-cell activation are distinct, confirming that NASC-seq simultaneously measures gene expression corresponding to two time points in single cells. Altogether, NASC-seq enables precise temporal monitoring of RNA synthesis at single-cell resolution during homoeostasis, perturbation responses and cellular differentiation.

SUBMITTER: Hendriks GJ

PROVIDER: S-EPMC6637240 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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NASC-seq monitors RNA synthesis in single cells.

Hendriks Gert-Jan GJ Jung Lisa A LA Larsson Anton J M AJM Lidschreiber Michael M Andersson Forsman Oscar O Lidschreiber Katja K Cramer Patrick P Sandberg Rickard R

Nature communications 20190717 1

Sequencing of newly synthesised RNA can monitor transcriptional dynamics with great sensitivity and high temporal resolution, but is currently restricted to populations of cells. Here, we develop new transcriptome alkylation-dependent single-cell RNA sequencing (NASC-seq), to monitor newly synthesised and pre-existing RNA simultaneously in single cells. We validate the method on pre-labelled RNA, and by demonstrating that more newly synthesised RNA was detected for genes with known high mRNA tur ...[more]

PMID: 31316066

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Project description:PurposeCorneal epithelial homeostasis is maintained by coordinated gene expression across distinct cell populations, but the gene regulatory programs underlying this cellular diversity remain to be characterized. Here we applied single-cell multi-omics analysis to delineate the gene regulatory profile of mouse corneal epithelial cells under normal homeostasis.MethodsSingle cells isolated from the cornea epithelium (with marginal conjunctiva) of adult mice were subjected to scRNA-seq and scATAC-seq using the 10×Genomics platform. Cell types were clustered by the graph-based visualization method uniform manifold approximation and projection and unbiased computational informatics analysis. The scRNA-seq and scATAC-seq datasets were integrated following the integration pipeline described in ArchR and Seurat.ResultsWe characterized diverse corneal epithelial cell types based on gene expression signatures and chromatin accessibility. We found that cell type-specific accessibility regions were mainly located at distal regions, suggesting essential roles of distal regulatory elements in determining corneal epithelial cell diversity. Trajectory analyses revealed a continuum of cell state transition and higher coordination between transcription factor (TF) motif accessibility and gene expression during corneal epithelial cell differentiation. By integrating transcriptomic and chromatin accessibility analysis, we identified cell type-specific and shared gene regulation programs. We also uncovered critical TFs driving corneal epithelial cell differentiation, such as nuclear factor I (NFI) family members, Rarg, Elf3. We found that nuclear factor-κB (NF-κB) family members were positive TFs in limbal cells and some superficial cells, but they were involved in regulating distinct biological processes.ConclusionsOur study presents a comprehensive gene regulatory landscape of mouse cornea epithelial cells, and provides valuable foundations for future investigation of corneal epithelial homeostasis in the context of cornea pathologies and regenerative medicine.

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NASC-seq monitors RNA synthesis in single cells.

Publications

NASC-seq monitors RNA synthesis in single cells.

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