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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\n                    <jats:italic>TMEM119<\/jats:italic>\n                    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.\n                  <\/jats:p>","DOI":"10.1038\/s41467-021-25871-2","type":"journal-article","created":{"date-parts":[[2021,10,12]],"date-time":"2021-10-12T08:05:45Z","timestamp":1634025945000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":65,"title":["High-throughput RNA sequencing of paraformaldehyde-fixed single cells"],"prefix":"10.1038","volume":"12","author":[{"given":"Hoang Van","family":"Phan","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michiel","family":"van Gent","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4460-9558","authenticated-orcid":false,"given":"Nir","family":"Drayman","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9468-3727","authenticated-orcid":false,"given":"Anindita","family":"Basu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michaela U.","family":"Gack","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1912-6020","authenticated-orcid":false,"given":"Sava\u015f","family":"Tay","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2021,9,24]]},"reference":[{"key":"25871_CR1","doi-asserted-by":"publisher","first-page":"eaah4573","DOI":"10.1126\/science.aah4573","volume":"356","author":"A-C Villani","year":"2017","unstructured":"Villani, A.-C. et al. Single-cell RNA-seq reveals new types of human blood dendritic cells, monocytes, and progenitors. Science 356, eaah4573 (2017).","journal-title":"Science"},{"key":"25871_CR2","doi-asserted-by":"publisher","first-page":"1131","DOI":"10.1038\/nsmb.2660","volume":"20","author":"L Yan","year":"2013","unstructured":"Yan, L. et al. Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells. Nat. Struct. Mol. Biol. 20, 1131 (2013).","journal-title":"Nat. Struct. Mol. Biol."},{"key":"25871_CR3","doi-asserted-by":"publisher","first-page":"1202","DOI":"10.1016\/j.cell.2015.05.002","volume":"161","author":"EZ Macosko","year":"2015","unstructured":"Macosko, E. Z. et al. Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. 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Cell fixation and preservation for droplet-based single-cell transcriptomics. BMC Biol. 15, 44 (2017).","journal-title":"BMC Biol."},{"key":"25871_CR10","doi-asserted-by":"publisher","DOI":"10.1186\/s12967-018-1578-4","volume":"16","author":"J Chen","year":"2018","unstructured":"Chen, J. et al. PBMC fixation and processing for chromium single-cell RNA sequencing. J. Transl. Med. 16, 198 (2018).","journal-title":"J. Transl. Med."},{"key":"25871_CR11","doi-asserted-by":"publisher","first-page":"432","DOI":"10.1002\/cyto.990130414","volume":"13","author":"AA Pollice","year":"1992","unstructured":"Pollice, A. A. et al. Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins. 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