Applying thiouracil tagging to mouse transcriptome analysis.

Area of Science:

• Molecular Biology
• Genomics
• Developmental Biology

Background:

• Transcriptional profiling is crucial for studying mouse models of development, physiology, and disease.
• Existing methods for cell-specific RNA analysis often involve cell isolation, which can induce transcriptional artifacts.
• There is a need for methods that directly assess active transcription in specific cell populations within complex tissues.

Purpose of the Study:

• To describe a protocol for mouse thiouracil tagging (TU tagging), a transcriptome analysis technology.
• To enable the isolation and analysis of cell type-specific RNA directly from complex tissues in vivo.
• To provide a method that avoids transcriptional changes associated with cell isolation and identifies actively transcribed RNAs.

Main Methods:

• In vivo covalent labeling of cell lineage-specific transcripts using 4-thiouracil in transgenic mice.
• Purification of TU-tagged RNA and subsequent library preparation for Illumina sequencing.
• Bioinformatics workflow for identifying cell type-enriched or differentially expressed genes.

Main Results:

• TU tagging allows for the direct examination of transcriptional regulation, unlike methods relying on ribosome or nuclei purification.
• The protocol enables the isolation of RNA from specific cell populations, circumventing cell isolation-induced transcriptional changes.
• Actively transcribed RNAs can be identified, distinguishing them from pre-existing transcripts.

Conclusions:

• TU tagging is a powerful and direct method for cell-specific transcriptional profiling in mice.
• The protocol is rapid, with tissue acquisition in 1 day, library generation in 2 days, and initial bioinformatics analysis in 1 day post-sequencing.
• This technology offers significant advantages for studying gene regulation in mouse development, physiology, and disease models.