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一种经济有效的人类皮肤单细胞 RNA 测序方案。

A cost-effective protocol for single-cell RNA sequencing of human skin.

机构信息

Graduate School of Health Sciences, Koç University, Istanbul, Türkiye.

Koç University Research Center for Translational Medicine, Koç University, Istanbul, Türkiye.

出版信息

Front Immunol. 2024 Oct 30;15:1393017. doi: 10.3389/fimmu.2024.1393017. eCollection 2024.

DOI:10.3389/fimmu.2024.1393017
PMID:39539550
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11557338/
Abstract

INTRODUCTION

Single-cell RNA sequencing (scRNAseq) and flow cytometry studies in skin are methodologically complex and costly, limiting their accessibility to researchers worldwide. Ideally, RNA and protein-based analyses should be performed on the same lesion to obtain more comprehensive data. However, current protocols generally focus on either scRNAseq or flow cytometry of healthy skin.

METHODS

We present a novel label-free sample multiplexing strategy, building on the souporcell algorithm, which enables scRNAseq analysis of paired blood and skin samples. Additionally, we provide detailed instructions for simultaneous flow cytometry analysis from the same sample, with necessary adaptations for both healthy and inflamed skin specimens.

RESULTS

This tissue multiplexing strategy mitigates technical batch effects and reduces costs by 2-4 times compared to existing protocols. We also demonstrate the effects of varying enzymatic incubation durations (1, 3, and 16 hours, with and without enzyme P) on flow cytometry outcomes. Comprehensive explanations of bioinformatic demultiplexing steps and a detailed step-by-step protocol of the entire experimental procedure are included.

DISCUSSION

The protocol outlined in this article will make scRNAseq and flow cytometry analysis of skin samples more accessible to researchers, especially those new to these techniques.

摘要

简介

单细胞 RNA 测序（scRNAseq）和皮肤流式细胞术研究方法复杂且昂贵，限制了其在全球研究人员中的可及性。理想情况下，应在同一病变部位进行基于 RNA 和蛋白质的分析，以获得更全面的数据。然而，目前的方案通常侧重于健康皮肤的 scRNAseq 或流式细胞术分析。

方法

我们提出了一种新颖的无标记样本多路复用策略，该策略基于 souporcell 算法，可实现配对血液和皮肤样本的 scRNAseq 分析。此外，我们还提供了从同一样本同时进行流式细胞术分析的详细说明，包括对健康和炎症皮肤标本的必要调整。

结果

与现有方案相比，这种组织多路复用策略可减轻技术批次效应并将成本降低 2-4 倍。我们还展示了不同酶孵育时间（1、3 和 16 小时，有和没有酶 P）对流式细胞术结果的影响。本文包括生物信息学解复用步骤的综合说明以及整个实验过程的详细分步协议。

讨论

本文概述的方案将使皮肤样本的 scRNAseq 和流式细胞术分析更容易被研究人员，特别是那些新技术的研究人员所接受。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/54f2/11557338/62e39ba672c5/fimmu-15-1393017-g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/54f2/11557338/bd25452d8573/fimmu-15-1393017-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/54f2/11557338/38589fb2d8c9/fimmu-15-1393017-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/54f2/11557338/dc2db82ac463/fimmu-15-1393017-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/54f2/11557338/82ef4bfe520a/fimmu-15-1393017-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/54f2/11557338/2e151a1a26db/fimmu-15-1393017-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/54f2/11557338/397bba7cb311/fimmu-15-1393017-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/54f2/11557338/62e39ba672c5/fimmu-15-1393017-g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/54f2/11557338/bd25452d8573/fimmu-15-1393017-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/54f2/11557338/38589fb2d8c9/fimmu-15-1393017-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/54f2/11557338/dc2db82ac463/fimmu-15-1393017-g003.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/54f2/11557338/2e151a1a26db/fimmu-15-1393017-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/54f2/11557338/397bba7cb311/fimmu-15-1393017-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/54f2/11557338/62e39ba672c5/fimmu-15-1393017-g007.jpg

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Single-cell profiles reveal distinctive immune response in atopic dermatitis in contrast to psoriasis.单细胞分析揭示了特应性皮炎与银屑病截然不同的免疫反应。
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Cross-tissue immune cell analysis reveals tissue-specific features in humans.跨组织免疫细胞分析揭示人类组织特异性特征。
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