从人类糖尿病纤维血管膜中分离单细胞,用于单细胞 RNA 测序。

IF 1 Q3 BIOLOGY Bio-protocol Pub Date : 2024-10-20 DOI:10.21769/BioProtoc.5096
Katia Corano Scheri, Thomas Tedeschi, Amani A Fawzi
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引用次数: 0

摘要

单细胞转录组分析已成为查询生理和病理条件下单细胞水平基因表达变化的强大工具。分析的质量在很大程度上取决于组织消化方案,目的是保留成千上万的单个活细胞,以便进行后续处理步骤和分析。已有多种使用不同酶消化组织的消化方案。苛刻的消化会损伤某些类型的细胞,但这可能是消化纤维化组织(如我们的实验条件)所必需的。在本文中,我们总结了一种 I 型胶原酶消化方案,用于制备从增殖性糖尿病视网膜病变(PDR)患者手术切除的纤维血管组织的单细胞悬液,以进行单细胞 RNA 测序(scRNA-Seq)分析。我们还详细介绍了我们在先前发表的一项研究中实施的数据分析。主要特点 - 从 PDR 患者分离的纤维血管膜中提取单细胞悬液。- 使用 RStudio 中的 Seurat 软件包进行单细胞 RNA 测序分析。- 轨迹分析或伪时间分析,研究特定细胞类型的(伪)时间轨迹。- 本方案要求使用 Illumina HiSEQ4000 仪器以及 R 和 RStudio 语言进行分析。
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Single Cell Isolation from Human Diabetic Fibrovascular Membranes for Single-Cell RNA Sequencing.

Single-cell transcriptomic analyses have emerged as very powerful tools to query the gene expression changes at the single-cell level in physiological and pathological conditions. The quality of the analysis is heavily dependent on tissue digestion protocols, with the goal of preserving thousands of single live cells to submit to the subsequent processing steps and analysis. Multiple digestion protocols that use different enzymes to digest the tissues have been described. Harsh digestion can damage certain cell types, but this might be required to digest especially fibrotic tissue as in our experimental condition. In this paper, we summarize a collagenase type I digestion protocol for preparing the single-cell suspension from fibrovascular tissues surgically removed from patients with proliferative diabetic retinopathy (PDR) for single-cell RNA sequencing (scRNA-Seq) analyses. We also provide a detailed description of the data analysis that we implemented in a previously published study. Key features • Single-cell suspension from fibrovascular membranes isolated from PDR patients. • Single-cell RNA sequencing analyses performed using Seurat package in RStudio. • Trajectory analyses or pseudotime analyses to study the trajectory over (pseudo)time of specific cell types. • This protocol requires Illumina HiSEQ4000 instrument and knowledge of R and RStudio language for the analyses.

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