Massively parallel and time-resolved RNA sequencing in single cells with scNT-seq

Abstract

Single-cell RNA sequencing offers snapshots of whole transcriptomes but obscures the temporal dynamics of RNA biogenesis and decay. Here we present single-cell new transcript tagging sequencing (scNT-Seq), a method for massively parallel analysis of newly-transcribed and pre-existing RNAs from the same cell. This droplet microfluidics-based method enables high-throughput chemical conversion on barcoded beads, efficiently marking metabolically labeled newly-transcribed RNAs with T-to-C substitutions. By simultaneously measuring new and old transcriptomes, scNT-Seq reveals neuronal subtype-specific gene regulatory networks and time-resolved RNA trajectories in response to brief (minutes) versus sustained (hours) neuronal activation. Integrating scNT-Seq with genetic perturbation reveals that DNA methylcytosine dioxygenases may inhibit stepwise transition from pluripotent embryonic stem cell state to intermediate and totipotent two-cell-embryo-like (2C-like) states by promoting global RNA biogenesis. Furthermore, pulse-chase scNT-Seq enables transcriptome-wide measurements of RNA stability in rare 2C-like cells. Time-resolved single-cell transcriptomic analysis thus opens new lines of inquiry regarding cell-type-specific RNA regulatory mechanisms..

CellRank for directed single-cell fate mapping

“…However, lineage relationships are lost in scRNA-seq due to its destructive nature—cells cannot be measured multiple times. Experimental approaches have been proposed to mitigate this problem; scRNA-seq can be combined with lineage tracing methods 2–5 that use heritable barcodes to follow clonal evolution over long time scales, and metabolic labeling 6–9 uses the ratio between nascent and mature RNA molecules to statistically link observed gene expression profiles over short time windows. Yet both strategies are mostly limited to in vitro applications, prompting the development of computational approaches to reconstruct pseudotime trajectories 1,10–16 .…”

Spatio-temporal mRNA dynamics in the early zebrafish embryo

“…3a). Several approaches for RNA labeling in single cells have been introduced recently 25–29,38 . However, these approaches are limited to cultured cells and have not been applied to live vertebrate embryos yet.…”

“…However, these approaches are limited to cultured cells and have not been applied to live vertebrate embryos yet. Furthermore, they are mostly plate-based and (with the exception of Qiu et al 29 ) not compatible with high-throughput single-cell RNA-seq by droplet microfluidics. In order to study embryonic development, and to also capture rare cell types such as germ cells, it was crucial to overcome these limitations.…”

“…However, a cell’s ‘future transcriptome’ can, within certain limits, be inferred from RNA sequencing data by counting the occurrence of intronic reads 24 . Moreover, recent methods have made considerable progress in directly measuring the transcriptional history of single cells in cell culture by introducing modified nucleotides into newly synthesized RNA 25−29 .…”

Simultaneous single cell measurements of intranuclear proteins and gene expression

“…We note that one drawback of inCITE-seq and related approaches is the need for high-quality antibodies. Future studies can combine inCITEseq with metabolic labeling 50, [92][93][94] , antibodies targeting phosphorylated forms of TFs, joint RNA and chromatin accessibility profiles 95 combined with spatial inference 96 Immunohistochemistry of mouse hippocampus. Mice were euthanized 2 hours after PBS or KA administration and perfused through the left ventricle with 20mL of PBS before gently removing the entire CNS.…”