Abstract
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.
Highlights
Single-cell transcriptomic studies that require intracellular protein staining, rare cell sorting, or inactivation of infectious pathogens are severely limited
The cells are individually lysed inside the droplets, and their mRNAs are captured by the oligonucleotides on the barcoded beads
FD-seq is useful for sequencing rare cell subpopulations that require intracellular staining and fluorescence-activated cell sorting (FACS)-enrichment, and for rendering infectious samples safe for handling
Summary
Single-cell transcriptomic studies that require intracellular protein staining, rare cell sorting, or inactivation of infectious pathogens are severely limited This is because current highthroughput 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. A high-throughput scRNA-seq method that combines well plate-based combinatorial indexing and the 10x platform, called scifi-RNA-seq[15], has been shown to work with formaldehyde-fixed single cells and single nuclei. Scifi-RNA-seq requires a separate reverse transcription step before droplet encapsulation, complicating the sample processing step Another method called inCITE-seq[16] has been developed for sequencing formaldehyde-fixed single nuclei with 10x.
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