High-throughput RNA sequencing of paraformaldehyde-fixed single cells

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.

Recent revelations and future directions using single-cell technologies in chronic lymphocytic leukemia

“…Because simultaneous sampling and the use of freshly harvested samples are generally not possible, the importance of standardized protocols across studies and collaborating centers cannot be overstated ( 125 ). Although the issue of initial PBMC storage and processing remains, the recently emerging possibility of using paraformaldehyde-fixed cells for scRNA-seq avoids the challenge of maintaining high cell viability through cryopreservation and transportation ( 126 , 127 ). Furthermore, if multi-omics is used, different applications may necessitate different sample preparation methods, which in turn requires careful coordination.…”

Advanced sequencing-based high-throughput and long-read single-cell transcriptome analysis

“…Phan et al developed FD-seq for PFA fixed cell subpopulation analysis. 52 To improve the RNA capture efficiency, PFA cross-linking was reversed by heating at 56 °C within the droplet to achieve the full release of RNA. The addition of protease K in the lysis buffer further improved the RNA yield.…”

Systematic benchmarking of imaging spatial transcriptomics platforms in FFPE tissues

“…A key historic limitation in the widespread use of iST methods with human clinical samples was the incompatibility of most methods with formalin-fixed, paraffin-embedded (FFPE) tissue samples [13, 14] . FFPE is the standard format for clinical sample preservation for pathology due to its ability to maintain tissue morphology and sample stability at room temperature for decades [15] .…”