Preparation of noninfectious scRNAseq samples from SARS-CoV-2-infected epithelial cells.
Preparation of noninfectious scRNAseq samples from SARS-CoV-2-infected epithelial cells.
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DOI：
10.1371/journal.pone.0281898
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发表时间：
2023
期刊：
影响因子：
3.7
通讯作者：
中科院分区：
文献类型：
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作者：
冠状病毒病 (COVID-19) 是由 SARS 冠状病毒 2 (SARS-CoV-2) 病毒引起的传染病。使用单细胞 RNA 测序 (scRNAseq) 等高通量方法进行直接评估、检测和定量分析对于了解宿主对 SARS-CoV-2 的反应至关重要。在实验室环境中研究 SARS-CoV-2 的一大障碍是需要在生物安全 3 级 (BSL-3) 控制下处理病毒感染的细胞培养物以及从中衍生的潜在感染性材料。然而，截至 2020 年，只有 190 个 BSL3 实验室设施在美国联邦选择代理计划中注册，并且其中只有一小部分配备了执行高通量分子测定所需的设备。在这里，我们描述了一种从 SARS-CoV-2 感染细胞中制备无害 RNA 样本的方法，该方法使得 scRNAseq 分析能够在 BSL2 设施中安全地进行，从而使更多的研究人员能够对 SARS-CoV-2 细胞进行分子测定。简而言之，我们用 SARS-CoV-2 感染非洲绿猴肾上皮细胞 (Vero-E6) 96 小时，胰蛋白酶解离细胞，并在单细胞悬浮液中用甲醇-丙酮灭活它们。使用组织培养感染剂量测定 (TCID50) 测试固定细胞是否存在感染性 SARS-CoV-2 病毒粒子，并使用流式细胞术测试活力。然后，我们在气液界面上分化的原代人肺上皮细胞上测试了解离和甲醇-丙酮灭活方法。最后，我们对所得细胞群进行了 scRNAseq 质量控制分析，以评估我们的病毒灭活和样品制备方案对产生的 cDNA 质量的影响。我们发现甲醇-丙酮可灭活SARS-CoV-2，固定肺上皮细胞，可用于获得非感染性、高质量的cDNA文库。这种方法使更多的研究人员能够更容易地在单细胞水平上研究 SARS-CoV-2 和相关的高遏制 RNA 病毒。
Coronavirus disease (COVID-19) is an infectious disease caused by the SARS coronavirus 2 (SARS-CoV-2) virus. Direct assessment, detection, and quantitative analysis using high throughput methods like single-cell RNA sequencing (scRNAseq) is imperative to understanding the host response to SARS-CoV-2. One barrier to studying SARS-CoV-2 in the laboratory setting is the requirement to process virus-infected cell cultures, and potentially infectious materials derived therefrom, under Biosafety Level 3 (BSL-3) containment. However, there are only 190 BSL3 laboratory facilities registered with the U.S. Federal Select Agent Program, as of 2020, and only a subset of these are outfitted with the equipment needed to perform high-throughput molecular assays. Here, we describe a method for preparing non-hazardous RNA samples from SARS-CoV-2 infected cells, that enables scRNAseq analyses to be conducted safely in a BSL2 facility–thereby making molecular assays of SARS-CoV-2 cells accessible to a much larger community of researchers. Briefly, we infected African green monkey kidney epithelial cells (Vero-E6) with SARS-CoV-2 for 96 hours, trypsin-dissociated the cells, and inactivated them with methanol-acetone in a single-cell suspension. Fixed cells were tested for the presence of infectious SARS-CoV-2 virions using the Tissue Culture Infectious Dose Assay (TCID50), and also tested for viability using flow cytometry. We then tested the dissociation and methanol-acetone inactivation method on primary human lung epithelial cells that had been differentiated on an air-liquid interface. Finally, we performed scRNAseq quality control analysis on the resulting cell populations to evaluate the effects of our virus inactivation and sample preparation protocol on the quality of the cDNA produced. We found that methanol-acetone inactivated SARS-CoV-2, fixed the lung epithelial cells, and could be used to obtain noninfectious, high-quality cDNA libraries. This methodology makes investigating SARS-CoV-2, and related high-containment RNA viruses at a single-cell level more accessible to an expanded community of researchers.
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影响因子：
3.5
作者：
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30.3
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评估SARS-COV-2的失活程序。
DOI：
10.1099/jgv.0.001539
发表时间：
2021-03
期刊：
The Journal of general virology
影响因子：
--
作者：
Auerswald H;Yann S;Dul S;In S;Dussart P;Martin NJ;Karlsson EA;Garcia-Rivera JA
通讯作者：
Garcia-Rivera JA