A two-step percoll gradient enables high-quality immune nuclei recovery from frozen melanoma for single-nucleus multiomics

Abstract Single-nucleus multiomic profiling of archived melanoma remains challenging due to melanin-associated debris and poor recovery of fragile immune nuclei. Here, we describe and validate a Two-Step Percoll (TSP) gradient method that enables efficient recovery of immune-cell nuclei from frozen human melanoma while supporting high-quality transcriptomic and chromatin accessibility profiling. Unlike FACS-based workflows, TSP requires no specialized sorting equipment, avoids mechanical stress on fragile nuclei, and effectively removes melanin-associated debris using only a standard benchtop centrifuge. TSP supports recovery of diverse tumor, stromal, and immune populations and high-quality paired snRNA-seq and snATAC-seq profiling from archived specimens. We validated the method across independent melanoma donors and show that it enables reproducible integrated transcriptomic and chromatin accessibility analysis from frozen clinical material. This simple, low-cost approach provides a practical workflow for single-nucleus multiomic profiling of frozen melanoma specimens.