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

The common octopus, Octopus vulgaris, is renowned for its advanced nervous system and complex behavior, yet its immune system remains poorly understood despite its relevance for health and pathogen resistance. Understanding octopus immunity is key to sustainable aquaculture and animal welfare. Single-cell RNA sequencing (scRNA-seq) provides high-resolution insights into immune cell diversity but requires viable single-cell suspensions, which are challenging in octopus hemocytes due to aggregation, high salinity, and chemical constraints. This study presents an optimized protocol for isolating circulating hemocytes and white body-resident hemocytes (WBH) from O. vulgaris. Hemocytes resuspended in Marine Antiaggregant Solution (MAS) maintained > 90% viability and structural integrity for at least 2 h post‑extraction, while WBH were dissociated using combined mechanical and enzymatic methods. Cell counting comparisons revealed poor correlation between LUNA-FL and flow cytometry, whereas Neubauer chamber counts aligned closely with cytometry data. Furthermore, a reduced-EDTA MAS medium (MAS low) proved compatible with 10 × Genomics chemistry, enabling successful GEM generation, reverse transcription, and cDNA library construction. Shallow sequencing confirmed recovery of high-quality transcriptomes and distinct hemocyte populations, demonstrating the feasibility of scRNA-seq in this non-model marine species. These findings provide the first methodological framework for applying scRNA-seq to octopus immune cells, enabling detailed analysis of hemocyte diversity and function. This pipeline establishes the foundation for investigating immune responses and adaptation and offers a valuable tool for extending single-cell applications to other marine invertebrates, enhancing immunity understanding of cephalopod immunity, and supporting animal welfare and sustainable aquaculture practices.