Fixed single-cell RNA sequencing for understanding virus infection and host response

ABSTRACT Single-cell transcriptomic studies that require intracellular protein staining, rare cell sorting, or inactivation of infectious pathogens are severely limited because current high-throughput RNA sequencing methods are incompatible with paraformaldehyde treatment, a common tissue and cell fixation and preservation technique. Here we present FD-seq, a high-throughput method for droplet-based RNA sequencing of paraformaldehyde-fixed, stained and sorted single-cells. We show that FD-seq preserves the mRNA integrity and relative abundances during fixation and subsequent cell retrieval. Furthermore, FD-seq detects a higher number of genes and transcripts than methanol fixation. We applied FD-seq to investigate two important questions in Virology. First, by analyzing a rare population of cells supporting lytic reactivation of the human tumor virus KSHV, we identified TMEM119 as a host factor that mediates viral reactivation. Second, we found that upon infection with the betacoronavirus OC43, which causes the common cold and is a close relative of SARS-CoV-2, pro-inflammatory pathways are primarily upregulated in lowly-infected cells that are exposed to the virus but fail to express high levels of viral genes. FD-seq thus enables integrating phenotypic with transcriptomic information in rare cell populations, and preserving and inactivating pathogenic samples that cannot be handled under regular biosafety measures..

Medienart:	Preprint

Erscheinungsjahr:	2021
Erschienen:	2021

Enthalten in:	bioRxiv.org - (2021) vom: 23. Jan. Zur Gesamtaufnahme - year:2021

Sprache:	Englisch

Beteiligte Personen:	Van Phan, Hoang [VerfasserIn] van Gent, Michiel [VerfasserIn] Drayman, Nir [VerfasserIn] Basu, Anindita [VerfasserIn] Gack, Michaela U. [VerfasserIn] Tay, Savaş [VerfasserIn]

Links:	Volltext [kostenfrei]

doi:	10.1101/2020.09.17.302232

funding:
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PPN (Katalog-ID):	XBI018801382