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

Jin-Hee Kim¹, Annalyssa N Long², Yumo Xie¹

¹Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, WA 98109, USA.

|May 15, 2026

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This study details an optimized protocol for single-nuclei RNA sequencing on snap-frozen mouse colon tissue. The method utilizes FLEX library preparation for high-quality molecular profiling of intact nuclei.

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.

Keywords:

Gene Expression Genomics RNAseq Single Cell

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Low-input Nucleus Isolation and Multiplexing with Barcoded Antibodies of Mouse Sympathetic Ganglia for Single-nucleus RNA Sequencing

Low-input Nucleus Isolation and Multiplexing with Barcoded Antibodies of Mouse Sympathetic Ganglia for Single-nucleus RNA Sequencing

Published on: March 23, 2022

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Last Updated: May 17, 2026

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

Published on: September 16, 2019

Low-input Nucleus Isolation and Multiplexing with Barcoded Antibodies of Mouse Sympathetic Ganglia for Single-nucleus RNA Sequencing

Low-input Nucleus Isolation and Multiplexing with Barcoded Antibodies of Mouse Sympathetic Ganglia for Single-nucleus RNA Sequencing

Published on: March 23, 2022

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.