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In addition to different in-house protocols, commercial whole-skin dissociation kits, primarily optimized for fibroblast harvest ( Kim et al., 2020), have often been used for skin dissociation. Longer digestion times may release greater cell numbers from tissues, while negatively impacting cell viability and potentially altering original cell transcriptomes. Additionally, the digestion times vary from 2 h to overnight digestion ( Kim et al., 2020). Published skin dissociation protocols differ in the number of recovered cells and their viability. Optimal dissociation conditions should finely balance the cell release from difficult-to-digest tissue while avoiding cellular damage during mechanical and enzymatic digestion. Minimizing the exposure of tissue to these stress factors can be crucial for capturing representative tissue cell heterogeneity for reliable scRNA-seq results. Different stress factors, such as mechanical damage, digestion temperature ( Denisenko et al., 2020 Slyper et al., 2020) and long enzymatic digestion times might skew cellular transcriptomes. Variations in tissue collection, storage and processing might influence the outcomes of downstream cell analyses including scRNA-seq outputs ( Denisenko et al., 2020 Slyper et al., 2020). These studies revealed the previously unrecognized cell heterogeneity of the skin, including the diversity of fibroblast ( Tabib et al., 2018 Solé-Boldo et al., 2020), keratinocyte ( Cheng et al., 2018 Wang et al., 2020), and immune cell subtypes ( He et al., 2020 Xu et al., 2021). Various skin dissociation protocols have been utilized in the last years to study human dermal pathologies at single cell resolution. Multiple tissue dissociation protocols have been published ( Kim et al., 2020 Dubois et al., 2021), aiming at isolating viable single cells from the tissue while preserving RNA integrity and cellular tissue composition.Ĭareful consideration of tissue digestion protocols is crucial, especially for hard-to-digest tissues, such as skin ( Jian et al., 2020), which require prolonged tissue dissociation. Recent advances in single-cell RNA-seq (scRNA-seq) technology have improved our understanding of human tissue complexity in health and disease. Overall, by enabling efficient cell isolation and comprehensive cell mapping, our skin dissociation-scRNA-seq workflow can greatly facilitate scRNA-seq discoveries across diverse human skin pathologies and ex vivo skin explant experimentations. The quality metrics of the generated scRNA-seq datasets were comparable between freshly dissociated and cultured skin. Furthermore, we effectively isolated highly viable single cells from ex vivo cultured skin biopsy fragments and generated a global single-cell map of the explanted human skin. We recapitulated not only the main cell populations of existing single-cell skin atlases, but also identified rare cell populations, such as mast cells. Our protocol enabled the isolation of a consistently high number of highly viable skin cells from small freshly dissociated punch skin biopsies, which we use for scRNA-seq studies. We present an optimized dissociation protocol for preparing high-quality skin cell suspensions for in-depth single-cell RNA-sequencing (scRNA-seq) analysis of fresh and cultured human skin. 6BioMed X Institute, Heidelberg, Germany.5Department of Dermatology, University of Zurich, University Hospital Zurich, Schlieren, Switzerland.4Department of Molecular Life Sciences and Swiss Institute of Bioinformatics, University of Zurich, Zurich, Switzerland.3Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.2Department of Rheumatology, University Medical Centre Ljubljana, Ljubljana, Slovenia.1Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zurich, University of Zurich, Zurich, Switzerland.Levesque 5 Oliver Distler 1 Žiga Rotar 2,3 Mark D. Edalat 1 Reto Gerber 1,4 Miranda Houtman 1 Muriel Elhai 1 Kristina Bürki 1 Ramon Staeger 5 Gaetana Restivo 5 Ramon Lang 5 Snezna Sodin-Semrl 2 Katja Lakota 2 Matija Tomšič 2,3 Mitchell P. Blaž Burja 1,2,3 † Dominique Paul 4 † Aizhan Tastanova 5 Sam G.
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