Extraction of nuclei from archived postmortem tissues for single-nucleus sequencing applications

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Single-cell and single-nucleus sequencing are powerful tools in biological research.
• Obtaining high-quality single-cell or single-nucleus suspensions is critical for these techniques.
• Archived and fragile tissues present challenges for preparing these suspensions, limiting their use in single-cell studies.

Purpose of the Study:

• To develop a fast and reliable protocol for preparing single-nucleus suspensions from various archived tissue types.
• To overcome the limitations of current methods that require specialized equipment or are time-consuming.
• To enable the application of single-nucleus sequencing technologies to challenging tissue samples.

Main Methods:

• A protocol involving tissue preparation and dissociation, nuclei extraction, and nuclei concentration assessment and capture was developed.
• The protocol utilizes standard laboratory equipment and takes only a few hours to complete.
• Key steps include nuclei extraction and concentration assessment, avoiding fluorescence-assisted nuclei sorting (FANS) or ultracentrifugation.

Main Results:

• The protocol reliably produces single-nucleus suspensions from multiple postmortem and archived tissue types.
• The prepared nuclei are compatible with downstream single-nucleus RNA sequencing (snRNA-seq) and assay for transposase-accessible chromatin (ATAC)-seq.
• High-quality snRNA-seq libraries are consistently generated using this method.

Conclusions:

• This protocol provides an accessible and efficient method for preparing single-nucleus suspensions from challenging tissue samples.
• It significantly broadens the scope of archived and fragile tissues that can be investigated using single-nucleus sequencing technologies.
• The method is suitable for routine use in graduate student laboratories and is compatible with 10X Genomics Chromium system workflows.