Development of a fixed skin biopsy protocol for single-cell RNA sequencing analysis of large samples

As part of the European SEAWave research consortium, we are studying the impact of 5G waves on the human body. We leadthe work package investigating the effects of 5G FR2 on human skin, while other groups conduct coordinated studies on cellsand animals. Participants, including healthy volunteers and patients with dermatoporosis, skin-cancer-prone syndromes, orinfl amed skin, are exposed to precise doses of 5G, resulting in 168 skin samples.
Objective
The large batch size necessitates standardization of the RNA extradition protocol that is not possible with traditional single-cell RNA sequencing (scRNA-seq)tools. For this reason, we developed a novel protocol for large-scale analysis of fi xed skinsamples.
Results
Our novel protocol addresses the challenge of dissociating intact cells from skin tissue. The samples are processed using therecently released Chromium Fixed RNA Profi ling Protocol, selected for its unbiased and sensitive nature, but it had never beenapplied to skin samples before. Optimal results were achieved by cutting biopsies in fi xation buffer, fi xing for 19 to 21 hours,dissociating in Liberase TL for one hour at 37°C and overnight at 4°C, followed by manual dissociation. Finally, intact cells aresorted using DAPI staining.
Conclusion
Using this refi ned protocol, we can detect all expected cell types in fi xed skin samples. The dermatological community standsto benefi t greatly from this robust new protocol, especially when dealing with numerous samples, something that will comemore and more the case in the near future.