Optimisation of single-nuclei isolation and RNA sequencing of parasitic nematodes

Abstract

Single-cell/nuclei transcriptomics has revolutionised our understanding of cell, tissue, and organismal biology in humans and model organisms across health and disease. However, for non-model species, reassessment and refinement of conventional methods is needed to address novel technical and biological challenges. Here, we evaluate three established nuclei isolation protocols and, building on these experiences, develop an optimised protocol for single-nuclei isolation of parasitic nematodes (SNIP). SNIP recovered 1.7- to 4.1-fold more nuclei than the three established protocols, and 3.2-fold more RNA per nucleus than the best-performing of these. We validated SNIP across macroscopic and microscopic life stages and both sexes of two phylogenetically distinct gastrointestinal nematodes, Haemonchus contortus and Trichuris muris, and used it to generate single-nuclei RNA-seq data revealing sex- and stage-specific gene expression and putative tissue-specific expression in both species. By enabling flexibility with frozen samples and consistent recovery across diverse parasitic nematode life stages, SNIP provides a practical foundation for building cell and nuclei atlases in other non-model species, toward advancing understanding from fundamental biology to infection and disease.