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A simple approach of nuclei isolation for single nucleus multiome sequencing
Yin Wang1, Di Ren2, Randy Kang3
1Department of Diabetes and Cancer Metabolism, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA 91010, USA; Division of Cardiology, Department of Medicine, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA 91010, USA.
We developed a fast, inexpensive nuclei isolation method called DFGC for frozen tissues. This technique improves single nucleus multiome sequencing (snMultiome-seq) data quality, especially for challenging samples like heart tissue.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Single nucleus multiome sequencing (snMultiome-seq) is crucial for understanding biological processes.
- Existing methods struggle to isolate high-quality nuclei from cryopreserved fibrous tissues, impacting data quality.
- Heart tissue presents particular challenges for nuclei isolation due to its fibrous nature.
Purpose of the Study:
- To develop a simple, inexpensive, and efficient protocol for isolating high-quality nuclei from frozen tissues.
- To evaluate the effectiveness of the new protocol compared to existing methods.
- To enhance the applicability of snMultiome-seq for challenging sample types.
Main Methods:
- Developed the douncer-filter-gradient-centrifugation (DFGC) protocol for nuclei isolation.
- Protocol involves mincing, douncing, filtration, and density gradient centrifugation.
- Compared DFGC with micro-beads and fluorescence-activated cell sorting (FACS) methods.
Main Results:
- The DFGC protocol is rapid, taking approximately 1.5 hours.
- DFGC yields superior nuclei quality for downstream snMultiome-seq applications.
- DFGC demonstrated preferred performance for both gene expression and chromatin accessibility data generation.
Conclusions:
- The DFGC method offers a significant improvement for nuclei isolation from frozen fibrous tissues.
- This protocol is expected to become a standard approach for high-quality snMultiome-seq.
- DFGC enhances the utility of snMultiome-seq for studying complex tissues like the heart.
