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

Area of Science:

• Molecular Biology
• Genomics
• Cell Biology

Background:

• Standard single-cell RNA sequencing provides static transcriptome snapshots, limiting insights into dynamic RNA processes.
• Understanding temporal RNA dynamics is crucial for deciphering cellular regulation and development.

Purpose of the Study:

• To develop a method for simultaneously analyzing newly transcribed and pre-existing mRNAs within individual cells.
• To enable time-resolved analysis of RNA dynamics at the single-cell level.

Main Methods:

• Developed single-cell metabolically labeled new RNA tagging sequencing (scNT-seq), a droplet microfluidics-based technique.
• scNT-seq chemically labels newly transcribed mRNAs with T-to-C substitutions on barcoded beads.
• Jointly profiled new and old transcriptomes in approximately 55,000 single cells.

Main Results:

• Revealed time-resolved transcription factor activities and cell-state trajectories during neuronal activation.
• Quantified RNA biogenesis and decay rates during transitions between pluripotent and 2C-like stem cell states.
• Identified DNA methylcytosine dioxygenase as an epigenetic barrier to the 2C-like cell state.

Conclusions:

• scNT-seq provides unprecedented temporal resolution for single-cell transcriptomic analysis.
• This method uncovers novel RNA regulatory mechanisms and cell-type-specific dynamics.
• Opens new avenues for investigating cellular regulation and developmental processes.