An improved comprehensive method for collecting single nuclei from shrimp ovaries.

Single-nucleus sequencing is a powerful tool for studying ovarian development. However, a standardized protocol for single-nucleus isolation in crustaceans is currently lacking. In this study, the key parameters for preparing nuclei suspension of Litopenaeus vannamei were optimized systematically for the first time, employing evaluation methods such as HE staining, trypan blue staining, scanning electron microscopy, 10 × Genomics quality control data, and t-SNE plots. The results showed that the optimal lysis efficiency of fresh ovarian tissue was achieved by manually grinding with 0.1% NP40 for 5 min, and the nuclei quality was ensured. Meanwhile, an iodixanol-assisted approach was used to reduce the mechanical damage to nuclei caused by centrifugation, significantly enhancing nuclear recovery. To capture as many nuclear types as possible, we employed a combination of high- and low-speed centrifugation, improving nuclear recovery and reducing data bias. Shrimp ovarian samples can be processed within 40 min or less. The single nuclei isolated from shrimp ovaries by the optimized method were uniformly round with clear boundaries, a significant reduction in clumping and debris, and minimal nuclear residue in the supernatant. The nuclear recovery rate increased from 4.78% to 40.56%. This study bridges the gap in single-cell research on the reproductive system of L. vannamei and provides new directions and insights for the preparation of nuclei suspension from complex tissue samples in the crustacean research.