Snail Hemocyte Isolation and Profiling

Pooled hemolymph (500µl) from 50 snails was recovered into 2ml tubes and mixed with 1.5ml of modified anticoagulant solution (98mM NaOH, 186mM NaCl, 1.7mM EDTA, 1.7mM citric acid) (13 (link)). Sample was passed through a 30µm pre-separation filter (Miltenyi Biotec) to eliminate cell aggregates and obtain a suspension of unique cells. Then hemocytes were counted using a Malassez chamber and cell viability was measured with trypan blue exclusion technique. Samples were then spin-down (2700g, 5min, 4°C) to pellet the hemocytes and re-suspended in 50µl of anticoagulant solution [30% of BGE medium (33 (link))] and 70% anticoagulant modified solution). To obtain a concentration of 1000 cells per microliter. Samples were then processed by MGX platform (IGH Montpellier, France) for scRNA droplet isolation (Chromium, 10X genomics) and RNA sequencing.