Protocol optimization to process mouse colon samples for single-nuclei RNA sequencing using FLEX library preparation

Area of Science:

• Molecular Biology
• Genomics
• Tissue Engineering

Background:

• Snap-freezing preserves clinical samples but damages cell membranes, hindering traditional single-cell analysis.
• Single-nuclei RNA sequencing (snRNA-seq) offers a solution by profiling intact nuclei from such samples.
• Efficient protocols are crucial for reliable snRNA-seq on preserved tissues.

Purpose of the Study:

• To present an optimized protocol for processing mouse colon tissue for snRNA-seq.
• To adapt existing methods for FLEX library preparation on fixed samples.
• To enable high-quality molecular profiling of snap-frozen clinical specimens.

Main Methods:

• Optimized protocol for mouse colon tissue preparation and dissociation.
• Nuclei isolation via filtration.
• Nuclei fixation for molecular preservation.
• Application of Chromium Fixed RNA Profiling (Gene Expression Flex) library preparation.

Main Results:

• Successful adaptation of snRNA-seq for snap-frozen mouse colon tissue.
• High-quality RNA yield and integrity from isolated nuclei.
• Detailed procedural steps for reproducible results.

Conclusions:

• The presented protocol enables effective snRNA-seq on snap-frozen colon tissue.
• This method preserves valuable molecular information from challenging clinical samples.
• Facilitates deeper understanding of tissue microenvironments using preserved specimens.