In summary, despite many technical advances, sequencing of PFA-fixed single cells continues be a complicated process with several shortcomings. Because inCITE-seq has only been demonstrated on formaldehyde-fixed cell nuclei, most of the mature mRNA transcripts that reside in the cytoplasm cannot be measured. Like scifi-RNA-seq, however, inCITE-seq requires laborious preprocessing of the samples. In contrast to scifi-RNA-seq, inCITE-seq performs cross-link reversal and reverse transcription inside the droplets. Another method called inCITE-seq 16 has been developed for sequencing formaldehyde-fixed single nuclei with 10x. However, scifi-RNA-seq requires a separate reverse transcription step before droplet encapsulation, thus complicating the sample processing step. Most recently, 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. A well plate-based method 5 was shown to be compatible with PFA-fixed cells, but the relatively low throughput nature of this method excludes its applicability from a wide range of problems that search for rare phenotypes in broad cellular populations. Important advances to scRNA-seq of PFA-fixed cells have recently been made. In many applications, however, PFA is preferred over methanol fixation due to the improved signal-to-background ratio in intracellular staining 11, 12, better preservation of intracellular structures’ integrity 13, or simply because methanol fixation does not produce a signal 14. High-throughput techniques like Drop-seq and 10x Chromium have been shown to be compatible with methanol-fixed cells 9, 10. Intracellular protein staining requires cell fixation, which is most commonly achieved with paraformaldehyde (PFA) or methanol fixation.
For example, Foxp3 is an intracellular marker of regulatory T cells 7, and Oct4 and Nanog are intracellular reprogramming markers of induced pluripotent stem cells 8. Many cell types require intracellular protein staining to be enriched. However, even with these high-throughput techniques, analyzing rare cell subpopulations remains a challenging task, often requiring protein-based enrichment for the subpopulation of interest before scRNA-seq 5, 6. Droplet-based scRNA-seq methods, such as Drop-seq 3 and 10x Chromium 4, are particularly powerful due to their high throughput: thousands of single cells can be analyzed in a single experiment. Single-cell RNA sequencing (scRNA-seq) has found many important biological applications, from the discovery of new cell types 1 to mapping the transcriptional landscape of human embryonic stem cells 2. FD-seq thus enables integrating phenotypic with transcriptomic information in rare cell subpopulations, and preserving and inactivating pathogenic samples. 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. 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. Furthermore, FD-seq can detect a higher number of genes and transcripts than methanol fixation. We show that FD-seq preserves the RNA integrity and relative gene expression levels after fixation and permeabilization. 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. 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. Single-cell transcriptomic studies that require intracellular protein staining, rare cell sorting, or inactivation of infectious pathogens are severely limited.
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