A simple approach of nuclei isolation for single nucleus multiome sequencing

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.