Optimized protocol for nuclei isolation from aquatic fish brain tissue for single-cell Genomic assays

Single-cell sequencing technology has become increasingly prominent in aquaculture for investigating complex biological processes, such as development, reproduction, and disease resistance in aquatic species like Nile tilapia, an economically important fish. This technology reveals cellular heterogeneity across various biological functions. However, traditional enzymatic dissociation methods often fail to generate high-quality, intact single-cell suspensions from complex brain tissue, limiting single-cell research in this area. This study aims to optimize the isolation of single-cell nuclei from tilapia ( Oreochromis niloticus ) brain tissue. We systematically fine-tuned the parameters of homogenization buffer, washing buffer, and centrifugation conditions, identifying the optimal method for nuclei isolation from tilapia brain. We developed a streamlined, rapid, and efficient protocol that eliminates the need for costly equipment, such as flow cytometry and ultracentrifugation. As a proof of principle, we successfully identified the cellular heterogeneity of tilapia brain tissue using single-nucleus RNA-seq analysis. The method we developed yields intact nuclei with minimal background contamination and a broad representation of cell types, making it a reliable tool for single-cell genomic assays in aquatic fish species.