Single-cell RNA sequencing studies requiring intracellular protein staining, rare-cell sorting, or pathogen inactivation are severely limited because current high-throughput methods are incompatible with paraformaldehyde treatment, a very common and simple tissue/cell fixation and preservation technique. Researchers from the University of Chicago present FD-seq, a high-throughput method for droplet-based RNA sequencing of paraformaldehyde-fixed, stained and sorted single-cells. The researchers used FD-seq to address two important questions in virology. First, by analyzing a rare population of cells supporting lytic reactivation of the human tumor virus KSHV, they identified TMEM119 as a host factor that mediates reactivation. Second, they studied the transcriptome of lung cells infected with the 2 coronavirus OC43, which causes the common cold and also serves as a safer model pathogen for SARS-CoV-2. The researchers found that pro-inflammatory pathways are primarily upregulated in abortively-infected or uninfected bystander cells, which are exposed to the virus but fail to express high level of viral genes.
FD-seq reveals pro-inflammatory bystander cells after coronavirus OC43 infection
(a-b) t-SNE plots with cells colored by (a) cluster identity or (b) sample type. (c) Bar plot showing the percentage of infected cells (defined as cells that expressed at least 1 viral transcript) of mock infected and MOI 1 sample. (d) Violin plot showing distribution of total viral transcript counts. (e) Violin plot showing the distribution of the percentage of total viral transcript by cluster identity. (f) Heatmap showing the relative expression level of each viral gene of each single cell in cluster 2. The blue line shows each cell’s percentage of total viral transcripts. (g) Violin plots showing the expression of four representative immune-related genes that are upregulated in cluster 0. (h) Bar plot showing the adjusted P-values of upregulated KEGG pathways in clusters 1 and 2. The vertical dashed line indicates P-value = 0.05.
FD-seq is suitable for characterizing rare cell populations of interest, for studying high-containment biological samples after inactivation, and for integrating intracellular phenotypic with transcriptomic information.