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ORIGINAL RESEARCH article

Front. Immunol.
Sec. Systems Immunology
Volume 15 - 2024 | doi: 10.3389/fimmu.2024.1393017
This article is part of the Research Topic Single Cell Technologies for the Interrogation of Immunological Disease Mechanisms View all 6 articles

A Cost-Effective Protocol for Single-Cell RNA Sequencing of Human Skin

Provisionally accepted
Saba Khoshbakht Saba Khoshbakht 1Özgür Albayrak Özgür Albayrak 1Ergün Tiryaki Ergün Tiryaki 2Orhan Ağcaoğlu Orhan Ağcaoğlu 3Ayse Oktem Ayse Oktem 4Gizem Pınar Sun Gizem Pınar Sun 5Elif Er Gulbezer Elif Er Gulbezer 6Sümeyre Seda Ertekin Sümeyre Seda Ertekin 7Ayşe Boyvat Ayşe Boyvat 4Atay Vural Atay Vural 1,8*Secil Vural Secil Vural 1,7*
  • 1 Koç University Research Center for Translational Medicine, Istanbul, Türkiye
  • 2 Koç University School of Medicine, Istanbul, Türkiye
  • 3 Department of Surgery, Koç University School of Medicine, Istanbul, Türkiye
  • 4 Department of Dermatology, Faculty of Medicine, Ankara University, Ankara, Ankara, Türkiye
  • 5 Department of Dermatology, Basaksehir Cam and Sakura City Hospital, Istanbul, Türkiye
  • 6 Department of Rheumatology, Koç University School of Medicine, Istanbul, Türkiye
  • 7 Department of Dermatology, School of Medicine, Koç University, Istanbul, Türkiye
  • 8 Department of Neurology, Koç University School of Medicine, Istanbul, Türkiye

The final, formatted version of the article will be published soon.

    Single-cell RNA sequencing (scRNAseq) and flow cytometry studies in skin are methodologically complex and expensive, limiting their accessibility to researchers worldwide. Ideally, RNA and protein-based analyses should be used complementarily on the same lesion to obtain more comprehensive information. However, existing protocols typically focus on either scRNAseq or flow cytometry analysis of healthy skin. In this paper, we introduce a novel protocol for scRNAseq analysis of immune cells in human skin that reduces costs by 2-4 times compared to existing protocols. Additionally, we provide detailed instructions for concurrent flow cytometry analysis from the same sample, outlining the necessary adaptations for analyzing both healthy and inflamed skin specimens. We explain a label-free sample multiplexing strategy that allows for the analysis of multiple samples and the essential demultiplexing steps. This strategy mitigates technical batch effects and reduces experimental costs. Furthermore, we demonstrate the impact of varying enzymatic incubation durations (1, 3, and 16 hours, with and without enzyme P) on flow cytometry results. In conclusion, we believe that the protocol presented in this article will make scRNAseq and flow cytometry analysis of skin samples more accessible to researchers new to these techniques.

    Keywords: Skin, Inflammation, Single cell RNA sequencing (scRNA), Flow Cytometry, souporcell, multiplexing, skin dissociation

    Received: 28 Feb 2024; Accepted: 08 Oct 2024.

    Copyright: © 2024 Khoshbakht, Albayrak, Tiryaki, Ağcaoğlu, Oktem, Pınar Sun, Er Gulbezer, Seda Ertekin, Boyvat, Vural and Vural. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence:
    Atay Vural, Department of Neurology, Koç University School of Medicine, Istanbul, Türkiye
    Secil Vural, Department of Dermatology, School of Medicine, Koç University, Istanbul, 34010, Türkiye

    Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.