当前位置: X-MOL 学术bioRxiv. Genom. › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
Fixed single-cell RNA sequencing for understanding virus infection and host response
bioRxiv - Genomics Pub Date : 2021-01-21 , DOI: 10.1101/2020.09.17.302232
Hoang Van Phan 1, 2 , Michiel van Gent 3, 4 , Nir Drayman 1, 2 , Anindita Basu 5 , Michaela U Gack 3, 4 , Savaş Tay 1, 2
Affiliation  

Single-cell transcriptomic studies that require intracellular protein staining, rare cell sorting, or inactivation of infectious pathogens are severely limited because current high-throughput RNA sequencing methods are incompatible with paraformaldehyde treatment, a common tissue and cell fixation and preservation technique. Here we present FD-seq, a high-throughput method for droplet-based RNA sequencing of paraformaldehyde-fixed, stained and sorted single-cells. We show that FD-seq preserves the mRNA integrity and relative abundances during fixation and subsequent cell retrieval. Furthermore, FD-seq detects a higher number of genes and transcripts than methanol fixation. We applied FD-seq to investigate two important questions in Virology. First, by analyzing a rare population of cells supporting lytic reactivation of the human tumor virus KSHV, we identified TMEM119 as a host factor that mediates viral reactivation. Second, we found that upon infection with the betacoronavirus OC43, which causes the common cold and is a close relative of SARS-CoV-2, pro-inflammatory pathways are primarily upregulated in lowly-infected 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 populations, and preserving and inactivating pathogenic samples that cannot be handled under regular biosafety measures.

中文翻译:

固定单细胞 RNA 测序以了解病毒感染和宿主反应

需要细胞内蛋白质染色、稀有细胞分选或感染性病原体灭活的单细胞转录组研究受到严重限制,因为目前的高通量 RNA 测序方法与多聚甲醛处理(一种常见的组织和细胞固定和保存技术)不兼容。在这里,我们介绍了 FD-seq,这是一种基于液滴的多聚甲醛固定、染色和分选单细胞 RNA 测序的高通量方法。我们表明 FD-seq 在固定和随后的细胞检索过程中保留了 mRNA 的完整性和相对丰度。此外,FD-seq 检测到比甲醇固定更多的基因和转录本。我们应用 FD-seq 来研究病毒学中的两个重要问题。首先,通过分析支持人类肿瘤病毒 KSHV 裂解再激活的稀有细胞群,我们将 TMEM119 鉴定为介导病毒再激活的宿主因子。其次,我们发现,在感染β冠状病毒 OC43(它会导致普通感冒并且是 SARS-CoV-2 的近亲)后,促炎途径主要在暴露于病毒但未能成功的低感染细胞中上调。表达高水平的病毒基因。因此,FD-seq 能够将表型与稀有细胞群中的转录组信息相结合,并保存和灭活在常规生物安全措施下无法处理的病原样本。促炎途径主要在暴露于病毒但不能表达高水平病毒基因的低感染细胞中上调。因此,FD-seq 能够将表型与稀有细胞群中的转录组信息相结合,并保存和灭活在常规生物安全措施下无法处理的病原样本。促炎途径主要在暴露于病毒但不能表达高水平病毒基因的低感染细胞中上调。因此,FD-seq 能够将表型与稀有细胞群中的转录组信息相结合,并保存和灭活在常规生物安全措施下无法处理的病原样本。
更新日期:2021-01-22
down
wechat
bug