Cargando…

Establishment of an Inactivation Method for Ebola Virus and SARS-CoV-2 Suitable for Downstream Sequencing of Low Cell Numbers

Technologies that facilitate the bulk sequencing of small numbers of cells as well as single-cell RNA sequencing (scRNA-seq) have aided greatly in the study of viruses as these analyses can be used to differentiate responses from infected versus bystander cells in complex systems, including in organ...

Descripción completa

Detalles Bibliográficos
Autores principales: Olejnik, Judith, Leon, Juliette, Michelson, Daniel, Chowdhary, Kaitavjeet, Galvan-Pena, Silvia, Benoist, Christophe, Mühlberger, Elke, Hume, Adam J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9958562/
https://www.ncbi.nlm.nih.gov/pubmed/36839614
http://dx.doi.org/10.3390/pathogens12020342
_version_ 1784895054314209280
author Olejnik, Judith
Leon, Juliette
Michelson, Daniel
Chowdhary, Kaitavjeet
Galvan-Pena, Silvia
Benoist, Christophe
Mühlberger, Elke
Hume, Adam J.
author_facet Olejnik, Judith
Leon, Juliette
Michelson, Daniel
Chowdhary, Kaitavjeet
Galvan-Pena, Silvia
Benoist, Christophe
Mühlberger, Elke
Hume, Adam J.
author_sort Olejnik, Judith
collection PubMed
description Technologies that facilitate the bulk sequencing of small numbers of cells as well as single-cell RNA sequencing (scRNA-seq) have aided greatly in the study of viruses as these analyses can be used to differentiate responses from infected versus bystander cells in complex systems, including in organoid or animal studies. While protocols for these analyses are typically developed with biosafety level 2 (BSL-2) considerations in mind, such analyses are equally useful for the study of viruses that require higher biosafety containment levels. Many of these workstreams, however, are not directly compatible with the more stringent biosafety regulations of BSL-3 and BSL-4 laboratories ensuring virus inactivation and must therefore be modified. Here we show that TCL buffer (Qiagen), which was developed for bulk sequencing of small numbers of cells and also facilitates scRNA-seq, inactivates both Ebola virus (EBOV) and SARS-CoV-2, BSL-4 and BSL-3 viruses, respectively. We show that additional heat treatment, necessary for the more stringent biosafety concerns for BSL-4-derived samples, was additionally sufficient to inactivate EBOV-containing samples. Critically, this heat treatment had minimal effects on extracted RNA quality and downstream sequencing results.
format Online
Article
Text
id pubmed-9958562
institution National Center for Biotechnology Information
language English
publishDate 2023
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-99585622023-02-26 Establishment of an Inactivation Method for Ebola Virus and SARS-CoV-2 Suitable for Downstream Sequencing of Low Cell Numbers Olejnik, Judith Leon, Juliette Michelson, Daniel Chowdhary, Kaitavjeet Galvan-Pena, Silvia Benoist, Christophe Mühlberger, Elke Hume, Adam J. Pathogens Article Technologies that facilitate the bulk sequencing of small numbers of cells as well as single-cell RNA sequencing (scRNA-seq) have aided greatly in the study of viruses as these analyses can be used to differentiate responses from infected versus bystander cells in complex systems, including in organoid or animal studies. While protocols for these analyses are typically developed with biosafety level 2 (BSL-2) considerations in mind, such analyses are equally useful for the study of viruses that require higher biosafety containment levels. Many of these workstreams, however, are not directly compatible with the more stringent biosafety regulations of BSL-3 and BSL-4 laboratories ensuring virus inactivation and must therefore be modified. Here we show that TCL buffer (Qiagen), which was developed for bulk sequencing of small numbers of cells and also facilitates scRNA-seq, inactivates both Ebola virus (EBOV) and SARS-CoV-2, BSL-4 and BSL-3 viruses, respectively. We show that additional heat treatment, necessary for the more stringent biosafety concerns for BSL-4-derived samples, was additionally sufficient to inactivate EBOV-containing samples. Critically, this heat treatment had minimal effects on extracted RNA quality and downstream sequencing results. MDPI 2023-02-17 /pmc/articles/PMC9958562/ /pubmed/36839614 http://dx.doi.org/10.3390/pathogens12020342 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Olejnik, Judith
Leon, Juliette
Michelson, Daniel
Chowdhary, Kaitavjeet
Galvan-Pena, Silvia
Benoist, Christophe
Mühlberger, Elke
Hume, Adam J.
Establishment of an Inactivation Method for Ebola Virus and SARS-CoV-2 Suitable for Downstream Sequencing of Low Cell Numbers
title Establishment of an Inactivation Method for Ebola Virus and SARS-CoV-2 Suitable for Downstream Sequencing of Low Cell Numbers
title_full Establishment of an Inactivation Method for Ebola Virus and SARS-CoV-2 Suitable for Downstream Sequencing of Low Cell Numbers
title_fullStr Establishment of an Inactivation Method for Ebola Virus and SARS-CoV-2 Suitable for Downstream Sequencing of Low Cell Numbers
title_full_unstemmed Establishment of an Inactivation Method for Ebola Virus and SARS-CoV-2 Suitable for Downstream Sequencing of Low Cell Numbers
title_short Establishment of an Inactivation Method for Ebola Virus and SARS-CoV-2 Suitable for Downstream Sequencing of Low Cell Numbers
title_sort establishment of an inactivation method for ebola virus and sars-cov-2 suitable for downstream sequencing of low cell numbers
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9958562/
https://www.ncbi.nlm.nih.gov/pubmed/36839614
http://dx.doi.org/10.3390/pathogens12020342
work_keys_str_mv AT olejnikjudith establishmentofaninactivationmethodforebolavirusandsarscov2suitablefordownstreamsequencingoflowcellnumbers
AT leonjuliette establishmentofaninactivationmethodforebolavirusandsarscov2suitablefordownstreamsequencingoflowcellnumbers
AT michelsondaniel establishmentofaninactivationmethodforebolavirusandsarscov2suitablefordownstreamsequencingoflowcellnumbers
AT chowdharykaitavjeet establishmentofaninactivationmethodforebolavirusandsarscov2suitablefordownstreamsequencingoflowcellnumbers
AT galvanpenasilvia establishmentofaninactivationmethodforebolavirusandsarscov2suitablefordownstreamsequencingoflowcellnumbers
AT benoistchristophe establishmentofaninactivationmethodforebolavirusandsarscov2suitablefordownstreamsequencingoflowcellnumbers
AT muhlbergerelke establishmentofaninactivationmethodforebolavirusandsarscov2suitablefordownstreamsequencingoflowcellnumbers
AT humeadamj establishmentofaninactivationmethodforebolavirusandsarscov2suitablefordownstreamsequencingoflowcellnumbers