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Preparation of noninfectious scRNAseq samples from SARS-CoV-2-infected epithelial cells

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...

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Autores principales: Osborn, Raven M., Leach, Justin, Zanche, Michelle, Ashton, John M., Chu, ChinYi, Thakar, Juilee, Dewhurst, Stephen, Rosenberger, Sonia, Pavelka, Martin, Pryhuber, Gloria S., Mariani, Thomas J., Anderson, Christopher S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9956660/
https://www.ncbi.nlm.nih.gov/pubmed/36827401
http://dx.doi.org/10.1371/journal.pone.0281898
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author Osborn, Raven M.
Leach, Justin
Zanche, Michelle
Ashton, John M.
Chu, ChinYi
Thakar, Juilee
Dewhurst, Stephen
Rosenberger, Sonia
Pavelka, Martin
Pryhuber, Gloria S.
Mariani, Thomas J.
Anderson, Christopher S.
author_facet Osborn, Raven M.
Leach, Justin
Zanche, Michelle
Ashton, John M.
Chu, ChinYi
Thakar, Juilee
Dewhurst, Stephen
Rosenberger, Sonia
Pavelka, Martin
Pryhuber, Gloria S.
Mariani, Thomas J.
Anderson, Christopher S.
author_sort Osborn, Raven M.
collection PubMed
description 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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spelling pubmed-99566602023-02-25 Preparation of noninfectious scRNAseq samples from SARS-CoV-2-infected epithelial cells Osborn, Raven M. Leach, Justin Zanche, Michelle Ashton, John M. Chu, ChinYi Thakar, Juilee Dewhurst, Stephen Rosenberger, Sonia Pavelka, Martin Pryhuber, Gloria S. Mariani, Thomas J. Anderson, Christopher S. PLoS One Lab Protocol 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. Public Library of Science 2023-02-24 /pmc/articles/PMC9956660/ /pubmed/36827401 http://dx.doi.org/10.1371/journal.pone.0281898 Text en © 2023 Osborn et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Lab Protocol
Osborn, Raven M.
Leach, Justin
Zanche, Michelle
Ashton, John M.
Chu, ChinYi
Thakar, Juilee
Dewhurst, Stephen
Rosenberger, Sonia
Pavelka, Martin
Pryhuber, Gloria S.
Mariani, Thomas J.
Anderson, Christopher S.
Preparation of noninfectious scRNAseq samples from SARS-CoV-2-infected epithelial cells
title Preparation of noninfectious scRNAseq samples from SARS-CoV-2-infected epithelial cells
title_full Preparation of noninfectious scRNAseq samples from SARS-CoV-2-infected epithelial cells
title_fullStr Preparation of noninfectious scRNAseq samples from SARS-CoV-2-infected epithelial cells
title_full_unstemmed Preparation of noninfectious scRNAseq samples from SARS-CoV-2-infected epithelial cells
title_short Preparation of noninfectious scRNAseq samples from SARS-CoV-2-infected epithelial cells
title_sort preparation of noninfectious scrnaseq samples from sars-cov-2-infected epithelial cells
topic Lab Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9956660/
https://www.ncbi.nlm.nih.gov/pubmed/36827401
http://dx.doi.org/10.1371/journal.pone.0281898
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