Li-BrU-seq: A Low-Input and Simplified Metabolic Labeling Method for Nascent RNA Sequencing

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• Transcriptional dynamics are crucial for biological processes like cell fate and stress responses.
• Nascent RNA sequencing is vital for studying gene expression but faces challenges like high cell input and cytotoxicity.
• Existing methods struggle with low-input samples and can introduce artifacts.

Purpose of the Study:

• To introduce Li-BrU-seq, an optimized 5-bromouridine (BrU)-based profiling strategy for low-input samples.
• To demonstrate Li-BrU-seq's superior performance compared to existing protocols.
• To provide a versatile and accessible platform for nascent RNA analysis.

Main Methods:

• Development of a systematically optimized 5-bromouridine (BrU)-based RNA profiling strategy.
• Streamlining the enrichment workflow for efficient low-input RNA analysis.
• Benchmarking Li-BrU-seq against previous protocols for specificity and sensitivity.

Main Results:

• Li-BrU-seq achieves higher enrichment specificity and sensitivity than existing methods.
• High-quality transcriptomic profiling is possible from low-input material (500 ng total RNA or ~25,000 cells).
• The method supports flexible temporal resolution without stress-induced artifacts, unlike 4sU-based methods.

Conclusions:

• Li-BrU-seq is an accessible and versatile platform for nascent RNA analysis.
• The method expands the scope of transcriptomic profiling to low-input, rare, and sensitive biological systems.
• Li-BrU-seq overcomes key limitations of current nascent RNA sequencing technologies.