An improved comprehensive method for collecting single nuclei from shrimp ovaries

Area of Science:

• Crustacean reproductive biology
• Single-cell genomics
• Molecular biology techniques

Background:

• Single-nucleus sequencing is vital for understanding ovarian development.
• A standardized protocol for crustacean single-nucleus isolation is currently unavailable.
• This limits single-cell research in commercially important species like Litopenaeus vannamei.

Purpose of the Study:

• To systematically optimize parameters for single-nucleus suspension preparation from Litopenaeus vannamei ovaries.
• To establish a robust and efficient protocol for isolating high-quality nuclei from crustacean ovarian tissue.
• To enhance nuclear recovery rates and reduce data bias in subsequent genomic analyses.

Main Methods:

• Optimization of ovarian tissue lysis using manual grinding and 0.1% NP40.
• Implementation of an iodixanol-assisted approach to minimize centrifugation-induced nuclear damage.
• Combination of high- and low-speed centrifugation for improved nuclear yield and diversity.
• Evaluation using HE staining, trypan blue staining, SEM, 10× Genomics QC, and t-SNE plots.

Main Results:

• Optimal lysis achieved by 5-minute manual grinding with 0.1% NP40, ensuring nuclei quality.
• Iodixanol-assisted method significantly enhanced nuclear recovery and reduced mechanical damage.
• Combined centrifugation improved nuclear recovery from 4.78% to 40.56% and reduced data bias.
• Isolated nuclei exhibited uniform shape, clear boundaries, reduced clumping, and minimal debris.

Conclusions:

• A novel, rapid (under 40 minutes), and effective protocol for single-nucleus isolation from shrimp ovaries was developed.
• This method significantly improves nuclear recovery and quality, addressing a critical gap in crustacean single-cell research.
• The optimized protocol provides valuable insights for preparing nuclei suspension from complex tissues in crustacean research.