High-throughput RNA sequencing of paraformaldehyde-fixed single cells
© 2021. The Author(s)..
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.
| Errataetall: | |
|---|---|
| Medienart: |
E-Artikel |
| Erscheinungsjahr: |
2021 |
|---|---|
| Erschienen: |
2021 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:12 |
|---|---|
| Enthalten in: |
Nature communications - 12(2021), 1 vom: 24. Sept., Seite 5636 |
| Sprache: |
Englisch |
|---|
| Beteiligte Personen: |
Phan, Hoang Van [VerfasserIn] |
|---|
| Links: |
|---|
| Themen: |
1HG84L3525 |
|---|
| Anmerkungen: |
Date Completed 26.10.2021 Date Revised 03.05.2023 published: Electronic UpdateOf: bioRxiv. 2021 Jan 21;:. - PMID 32995793 Citation Status MEDLINE |
|---|
| doi: |
10.1038/s41467-021-25871-2 |
|---|
| funding: |
|
|---|---|
| Förderinstitution / Projekttitel: |
|
| PPN (Katalog-ID): |
NLM33103106X |
|---|
| LEADER | 01000naa a22002652 4500 | ||
|---|---|---|---|
| 001 | NLM33103106X | ||
| 003 | DE-627 | ||
| 005 | 20231225212554.0 | ||
| 007 | cr uuu---uuuuu | ||
| 008 | 231225s2021 xx |||||o 00| ||eng c | ||
| 024 | 7 | |a 10.1038/s41467-021-25871-2 |2 doi | |
| 028 | 5 | 2 | |a pubmed24n1103.xml |
| 035 | |a (DE-627)NLM33103106X | ||
| 035 | |a (NLM)34561439 | ||
| 040 | |a DE-627 |b ger |c DE-627 |e rakwb | ||
| 041 | |a eng | ||
| 100 | 1 | |a Phan, Hoang Van |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a High-throughput RNA sequencing of paraformaldehyde-fixed single cells |
| 264 | 1 | |c 2021 | |
| 336 | |a Text |b txt |2 rdacontent | ||
| 337 | |a ƒaComputermedien |b c |2 rdamedia | ||
| 338 | |a ƒa Online-Ressource |b cr |2 rdacarrier | ||
| 500 | |a Date Completed 26.10.2021 | ||
| 500 | |a Date Revised 03.05.2023 | ||
| 500 | |a published: Electronic | ||
| 500 | |a UpdateOf: bioRxiv. 2021 Jan 21;:. - PMID 32995793 | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a © 2021. The Author(s). | ||
| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, N.I.H., Extramural | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 7 | |a Polymers |2 NLM | |
| 650 | 7 | |a Formaldehyde |2 NLM | |
| 650 | 7 | |a 1HG84L3525 |2 NLM | |
| 650 | 7 | |a RNA |2 NLM | |
| 650 | 7 | |a 63231-63-0 |2 NLM | |
| 650 | 7 | |a paraform |2 NLM | |
| 650 | 7 | |a Y19UC83H8E |2 NLM | |
| 700 | 1 | |a van Gent, Michiel |e verfasserin |4 aut | |
| 700 | 1 | |a Drayman, Nir |e verfasserin |4 aut | |
| 700 | 1 | |a Basu, Anindita |e verfasserin |4 aut | |
| 700 | 1 | |a Gack, Michaela U |e verfasserin |4 aut | |
| 700 | 1 | |a Tay, Savaş |e verfasserin |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t Nature communications |d 2010 |g 12(2021), 1 vom: 24. Sept., Seite 5636 |w (DE-627)NLM199274525 |x 2041-1723 |7 nnns |
| 773 | 1 | 8 | |g volume:12 |g year:2021 |g number:1 |g day:24 |g month:09 |g pages:5636 |
| 856 | 4 | 0 | |u http://dx.doi.org/10.1038/s41467-021-25871-2 |3 Volltext |
| 912 | |a GBV_USEFLAG_A | ||
| 912 | |a GBV_NLM | ||
| 951 | |a AR | ||
| 952 | |d 12 |j 2021 |e 1 |b 24 |c 09 |h 5636 | ||