Heat Inactivation of Nipah Virus for Downstream Single-Cell RNA Sequencing Does Not Interfere with Sample Quality

Adam J. Hume, Judith Olejnik, Mitchell R. White, Jessie Huang, Jacquelyn Turcinovic, Baylee Heiden, Pushpinder S. Bawa, Christopher J. Williams, Nickolas G. Gorham, Yuriy O. Alekseyev, John H. Connor, Darrell N. Kotton, Elke Mühlberger

Research output: Contribution to journalArticlepeer-review

Abstract

Single-cell RNA sequencing (scRNA-seq) technologies are instrumental to improving our understanding of virus–host interactions in cell culture infection studies and complex biological systems because they allow separating the transcriptional signatures of infected versus non-infected bystander cells. A drawback of using biosafety level (BSL) 4 pathogens is that protocols are typically developed without consideration of virus inactivation during the procedure. To ensure complete inactivation of virus-containing samples for downstream analyses, an adaptation of the workflow is needed. Focusing on a commercially available microfluidic partitioning scRNA-seq platform to prepare samples for scRNA-seq, we tested various chemical and physical components of the platform for their ability to inactivate Nipah virus (NiV), a BSL-4 pathogen that belongs to the group of nonsegmented negative-sense RNA viruses. The only step of the standard protocol that led to NiV inactivation was a 5 min incubation at 85 °C. To comply with the more stringent biosafety requirements for BSL-4-derived samples, we included an additional heat step after cDNA synthesis. This step alone was sufficient to inactivate NiV-containing samples, adding to the necessary inactivation redundancy. Importantly, the additional heat step did not affect sample quality or downstream scRNA-seq results.

Original languageEnglish (US)
Article number62
JournalPathogens
Volume13
Issue number1
DOIs
StatePublished - Jan 2024
Externally publishedYes

Keywords

  • 10x Genomics
  • BSL-4 pathogens
  • heat inactivation
  • iPSC-derived alveolar type 2 cells
  • Nipah virus
  • nonsegmented negative sense RNA viruses
  • scRNA-seq
  • single-cell RNA sequencing
  • VSV

ASJC Scopus subject areas

  • Immunology and Allergy
  • Molecular Biology
  • General Immunology and Microbiology
  • Microbiology (medical)
  • Infectious Diseases

Fingerprint

Dive into the research topics of 'Heat Inactivation of Nipah Virus for Downstream Single-Cell RNA Sequencing Does Not Interfere with Sample Quality'. Together they form a unique fingerprint.

Cite this