tetano
Editor, Senior Moderator
PLoS One
. 2023 Feb 24;18(2):e0281898.
doi: 10.1371/journal.pone.0281898. eCollection 2023.
Preparation of noninfectious scRNAseq samples from SARS-CoV-2-infected epithelial cells
Raven M Osborn[SUP] 1 2 3 [/SUP], Justin Leach[SUP] 3 [/SUP], Michelle Zanche[SUP] 4 [/SUP], John M Ashton[SUP] 4 [/SUP], ChinYi Chu[SUP] 5 [/SUP], Juilee Thakar[SUP] 1 2 3 6 7 8 [/SUP], Stephen Dewhurst[SUP] 2 3 [/SUP], Sonia Rosenberger[SUP] 9 10 [/SUP], Martin Pavelka[SUP] 3 10 [/SUP], Gloria S Pryhuber[SUP] 5 11 [/SUP], Thomas J Mariani[SUP] 5 [/SUP], Christopher S Anderson[SUP] 5 12 [/SUP]
Affiliations
Abstract
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.
Raven M Osborn[SUP] 1 2 3 [/SUP], Justin Leach[SUP] 3 [/SUP], Michelle Zanche[SUP] 4 [/SUP], John M Ashton[SUP] 4 [/SUP], ChinYi Chu[SUP] 5 [/SUP], Juilee Thakar[SUP] 1 2 3 6 7 8 [/SUP], Stephen Dewhurst[SUP] 2 3 [/SUP], Sonia Rosenberger[SUP] 9 10 [/SUP], Martin Pavelka[SUP] 3 10 [/SUP], Gloria S Pryhuber[SUP] 5 11 [/SUP], Thomas J Mariani[SUP] 5 [/SUP], Christopher S Anderson[SUP] 5 12 [/SUP]
Affiliations
Abstract
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.
Raven M Osborn[SUP] 1 2 3 [/SUP], Justin Leach[SUP] 3 [/SUP], Michelle Zanche[SUP] 4 [/SUP], John M Ashton[SUP] 4 [/SUP], ChinYi Chu[SUP] 5 [/SUP], Juilee Thakar[SUP] 1 2 3 6 7 8 [/SUP], Stephen Dewhurst[SUP] 2 3 [/SUP], Sonia Rosenberger[SUP] 9 10 [/SUP], Martin Pavelka[SUP] 3 10 [/SUP], Gloria S Pryhuber[SUP] 5 11 [/SUP], Thomas J Mariani[SUP] 5 [/SUP], Christopher S Anderson[SUP] 5 12 [/SUP]
Affiliations
Abstract
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.
. 2023 Feb 24;18(2):e0281898.
doi: 10.1371/journal.pone.0281898. eCollection 2023.
Preparation of noninfectious scRNAseq samples from SARS-CoV-2-infected epithelial cells
Raven M Osborn[SUP] 1 2 3 [/SUP], Justin Leach[SUP] 3 [/SUP], Michelle Zanche[SUP] 4 [/SUP], John M Ashton[SUP] 4 [/SUP], ChinYi Chu[SUP] 5 [/SUP], Juilee Thakar[SUP] 1 2 3 6 7 8 [/SUP], Stephen Dewhurst[SUP] 2 3 [/SUP], Sonia Rosenberger[SUP] 9 10 [/SUP], Martin Pavelka[SUP] 3 10 [/SUP], Gloria S Pryhuber[SUP] 5 11 [/SUP], Thomas J Mariani[SUP] 5 [/SUP], Christopher S Anderson[SUP] 5 12 [/SUP]
Affiliations
- PMID: 36827401
- DOI: 10.1371/journal.pone.0281898
Abstract
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.
Raven M Osborn[SUP] 1 2 3 [/SUP], Justin Leach[SUP] 3 [/SUP], Michelle Zanche[SUP] 4 [/SUP], John M Ashton[SUP] 4 [/SUP], ChinYi Chu[SUP] 5 [/SUP], Juilee Thakar[SUP] 1 2 3 6 7 8 [/SUP], Stephen Dewhurst[SUP] 2 3 [/SUP], Sonia Rosenberger[SUP] 9 10 [/SUP], Martin Pavelka[SUP] 3 10 [/SUP], Gloria S Pryhuber[SUP] 5 11 [/SUP], Thomas J Mariani[SUP] 5 [/SUP], Christopher S Anderson[SUP] 5 12 [/SUP]
Affiliations
- PMID: 36827401
- DOI: 10.1371/journal.pone.0281898
Abstract
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.
Raven M Osborn[SUP] 1 2 3 [/SUP], Justin Leach[SUP] 3 [/SUP], Michelle Zanche[SUP] 4 [/SUP], John M Ashton[SUP] 4 [/SUP], ChinYi Chu[SUP] 5 [/SUP], Juilee Thakar[SUP] 1 2 3 6 7 8 [/SUP], Stephen Dewhurst[SUP] 2 3 [/SUP], Sonia Rosenberger[SUP] 9 10 [/SUP], Martin Pavelka[SUP] 3 10 [/SUP], Gloria S Pryhuber[SUP] 5 11 [/SUP], Thomas J Mariani[SUP] 5 [/SUP], Christopher S Anderson[SUP] 5 12 [/SUP]
Affiliations
- PMID: 36827401
- DOI: 10.1371/journal.pone.0281898
Abstract
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.