Comprehensive guide for optimizing octopus immune cell preparation to enhance single cell RNA sequencing success

Area of Science:

• Marine Biology
• Immunology
• Genomics

Background:

• The immune system of the common octopus (Octopus vulgaris) is poorly understood, hindering efforts in sustainable aquaculture and animal welfare.
• Single-cell RNA sequencing (scRNA-seq) offers high-resolution insights into immune cell diversity but faces challenges with octopus hemocytes due to aggregation and environmental constraints.

Purpose of the Study:

• To develop and optimize a protocol for isolating viable octopus hemocytes for scRNA-seq analysis.
• To establish a methodological framework for investigating octopus immune cell diversity and function.

Main Methods:

• Optimized protocol for isolating circulating hemocytes and white body-resident hemocytes (WBH) from O. vulgaris.
• Utilized Marine Antiaggregant Solution (MAS) for hemocyte preservation and mechanical/enzymatic dissociation for WBH.
• Validated cell counting methods and adapted chemistry for scRNA-seq library preparation.

Main Results:

• Achieved >90% hemocyte viability and structural integrity for at least 2 hours post-extraction using MAS.
• Demonstrated successful single-cell RNA sequencing library construction and recovery of high-quality transcriptomes.
• Confirmed distinct hemocyte populations, validating the feasibility of scRNA-seq in O. vulgaris.

Conclusions:

• Established the first methodological framework for scRNA-seq in octopus immune cells, enabling detailed hemocyte analysis.
• The developed pipeline supports research into octopus immune responses and adaptation.
• Provides a valuable tool for extending single-cell applications to other marine invertebrates, benefiting cephalopod immunity research, animal welfare, and aquaculture.