TORNADO-seq: A Protocol for High-Throughput Targeted RNA-seq-Based Drug Screening in Organoids

Area of Science:

• Biotechnology
• Genomics
• Pharmacology

Background:

• Organoids, 3D ex vivo cell aggregates, closely mimic tissue homeostasis and offer advantages over 2D cell lines and mouse models for drug screening and translational research.
• Despite organoids' potential, established RNA-sequencing-based drug screening platforms are lacking.
• The field of organoid research is rapidly advancing with continuous development of new manipulation techniques.

Purpose of the Study:

• To provide a detailed protocol for TORNADO-seq (Targeted RNA-seq-based drug-screening method in organoids).
• To establish a cost-effective and sensitive RNA-seq-based drug screening platform for organoids.
• To enable classification and grouping of drugs based on complex phenotypic readouts, even without prior knowledge of their mechanisms of action.

Main Methods:

• Development and detailed protocol for TORNADO-seq, a targeted RNA-sequencing approach for organoid drug screening.
• Utilizing complex phenotypic analysis with a large number of selected readouts.
• Combining cost-effectiveness with sensitive detection of multiple cell identities, signaling pathways, and key phenotypic drivers.

Main Results:

• TORNADO-seq allows for direct classification and grouping of drugs based on complex phenotypes.
• The method is sensitive in detecting multiple cell identities and signaling pathways within organoids.
• It enables the identification of key drivers of cellular phenotypes relevant to drug action.

Conclusions:

• TORNADO-seq offers a novel, cost-effective, and sensitive RNA-seq-based drug screening platform for organoids.
• This high-content screening method provides unique insights into drug effects by analyzing complex phenotypes.
• TORNADO-seq can be applied to various organoid systems, yielding information unobtainable through other methods.