High-throughput RNA sequencing of paraformaldehyde-fixed single cells

Hoang Van Phan, Michiel van Gent, Nir Drayman, Anindita Basu, Michaela U. Gack, Savaş Tay*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

27 Citations (Scopus)


Single-cell transcriptomic studies that require intracellular protein staining, rare cell sorting, or inactivation of infectious pathogens are severely limited. This is because current high-throughput single-cell RNA sequencing methods are either incompatible with or necessitate laborious sample preprocessing for paraformaldehyde treatment, a common tissue and cell fixation and preservation technique. Here we present FD-seq (Fixed Droplet RNA sequencing), a high-throughput method for droplet-based RNA sequencing of paraformaldehyde-fixed, permeabilized and sorted single cells. We show that FD-seq preserves the RNA integrity and relative gene expression levels after fixation and permeabilization. Furthermore, FD-seq can detect a higher number of genes and transcripts than methanol fixation. We first apply FD-seq to analyze a rare subpopulation of cells supporting lytic reactivation of the human tumor virus KSHV, and identify TMEM119 as a potential host factor that mediates viral reactivation. Second, we find that infection with the human betacoronavirus OC43 leads to upregulation of pro-inflammatory pathways in 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 subpopulations, and preserving and inactivating pathogenic samples.

Original languageEnglish
Article number5636
JournalNature Communications
Issue number1
Publication statusPublished - 24 Sept 2021
Externally publishedYes

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