A simple approach of nuclei isolation for single nucleus multiome sequencing.
Publication Year:
2025
PubMed ID:
40849079
Public Summary:
In recent years, a new technology called single-nucleus multiome sequencing (snMultiome-seq) has allowed scientists to study what individual cells are doing by looking at both their gene activity and how their DNA is organized. However, this powerful technique has a big challenge: it is very hard to get good-quality cell nuclei from tissues that have been frozen for a long time—especially tough, fibrous tissues like the heart. To
solve this problem, we developed an easy, low-cost method to collect nuclei from frozen tissues. We call it the douncer-filter-gradient-centrifugation (DFGC) method. The whole process takes about 1.5 hours and includes four simple steps: cutting the tissue, gently breaking it apart, filtering it, and then separating the nuclei using a special spinning process. We tested the DFGC method against two commonly used techniques—
microbeads and FACS—and found that DFGC works better for producing high-quality nuclei. These nuclei perform well for advanced tests that measure gene activity and DNA accessibility. In short, the DFGC method is a simple and effective way to prepare frozen, fibrous tissues—like heart samples—for modern sequencing technologies.
Scientific Abstract:
The emergence of single nucleus multiome sequencing (snMultiome-seq) technology has greatly advanced our understanding of various biological processes. However, existing experimental protocols fail to isolate high-quality nuclei from cryopreserved fibrous tissues, such as the heart, leading to low-quality downstream sequencing data. Here, we develop a simple and inexpensive approach for nuclei isolation from frozen tissues, named douncer-filter-gradient-centrifugation (DFGC). This protocol takes approximately 1.5 h to complete, including mincing (1 min), douncing (3 min), filtration (20 min), and density gradient centrifugation (40 min). To evaluate the effectiveness of the DFGC approach, we compare it with two commonly used methods for nuclei isolation - micro-beads and fluorescence-activated cell sorting (FACS). We demonstrate that the DFGC method performs in a preferred manner for the generation of both single nucleus gene expression and chromatin transposase accessibility data. We anticipate the DFGC method to be a mainstream approach for high-quality nuclei isolation in snMultiome-seq.
