Optimized nucleus isolation protocol from frozen mouse tissues for single nucleus RNA sequencing application.

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Single cell RNA sequencing (scRNA-seq) has revolutionized biological research.
• Limitations exist, particularly with fresh tissue accessibility and sample integrity during dissociation.
• Single nucleus RNA sequencing (snRNA-seq) offers an alternative using frozen samples.

Purpose of the Study:

• To develop an efficient and adaptable nucleus isolation protocol for frozen complex tissues.
• To enable snRNA-seq applications on samples unsuitable for traditional methods.

Main Methods:

• Developed a protocol combining enzymatic and manual dissociation for nucleus isolation from frozen murine placenta and pancreas.
• Utilized standard laboratory reagents and equipment, avoiding commercial kits.
• Nuclei were sorted using flow cytometry prior to sequencing.

Main Results:

• The protocol successfully isolated nuclei from frozen placenta and pancreas.
• The method is easy to implement, cost-effective, and adaptable to various tissues and species.
• Validated efficiency for snRNA-seq on complex samples resistant to standard lysis.

Conclusions:

• This novel nucleus isolation protocol overcomes limitations of scRNA-seq for frozen and sensitive tissues.
• It expands the applicability of snRNA-seq to retrospective studies and challenging sample types like fibrotic or fatty tissues.
• Facilitates broader research in genomics and molecular biology.