Nuclei isolation from frozen heart tissues through an easy-to-operating, prompt, and inexpensive method for single-nucleus Multiome-seq
Existing protocols are difficult to obtain high-quality sequencing data for isolating nuclei from long-term cryopreserved frozen tissues, especially such as hearts that contain rich fibrous connective tissue for single-nucleus multiome sequencing (snMultiome-seq, including snRNA-seq and snATAC-seq for the same nucleus). Here, we optimize some current methods for isolating nuclei from frozen tissues and describe an easy-to-operating, prompt, and inexpensive method for obtaining high-quality single-nucleus sequencing data, which is named as Douncer-Filter-Gradient-Centrifugation (DFGC). This protocol takes about 1.5h to complete, including mincing (1min), douncing (3min), filtering (20min) and density gradient centrifugating (40min), depending on how many samples are done at the same time, and does not require special reagents and instruments, so it is versatile and economical for most laboratories. We selected two commonly used isolating nuclei methods, microBeads- and FACS-, as controls and all analysis processes are carried out in accordance with unified standardization, including nucleus morphology, library construction, library quality inspection, data parameters, clustering, cell-type identification, and gene expression distribution. This protocol provides a step-by-step guide to nuclei isolating from frozen heart tissues to generate high-quality single nucleus gene expression and chromatin transposase accessibility sequencing data for the same nucleus. Overall design: We isolated nuclei from the frozen heart tissues of C57BL/6J mice with three different protocols to examine the quality differences.