Gene Expression
Nebulas
Gene Expression NebulasSummary: Viral infection is usually studied at the population level by averaging over millions of cells. However, infection at the single-cell level is highly heterogeneous, where most infected cells give rise to none or few viral progeny while some cells produce thousands. Analysis of HSV-1 infection by population averaged measurements has taught us a lot about the course of viral infection, but has also produced contradictory results, such as the concurrent activation and inhibition of type I interferon signaling during infection. Here, we combine live-cell imaging and single-cell RNA sequencing to characterize viral and host transcriptional heterogeneity during HSV-1 infection of primary human cells. We find extreme variability in the level of viral gene expression among individually infected cells and show that they cluster into transcriptionally distinct sub-populations. We find that anti-viral signaling is initiated in a rare group of abortively infected cells, while highly infected cells undergo cellular reprogramming to an embryonic-like transcriptional state. This reprogramming includes the re-localization of b-catenin into the host nucleus and viral replication compartments and is required for late viral gene expression and progeny production. These findings uncover the transcriptional differences in cells with variable infection outcomes and shed new light on the manipulation of host pathways by HSV-1.
Overall Design: HDFn cells mock-infeted, infected with wt HSV-1 or dICP0 HSV-1 for 5 hours. drop-seq data for single-cell RNA-sequncing as well as bulk RNA-seq of sorted cell population (ICP4 positive or negative in each experimet, in duplicates)
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| Growth Protocol: | - |
| Treatment Protocol: | Cells were infected with HSV-1 for 1 hr at room temperature in miminal volume, washed and incubated for 5 hours. For sorting, cells were detached and sorted on a FACSAria Fusion |
| Extract Protocol: | For bulk RNA Qiagen miniprep RNA extractio colum were used. For drop-seq we followed the drop-seq protocol in Macosko et al 2015.Standard Illuimina protocol for bulk RNA-seq, according to Macosko et al. 2015 for drop-seq |
| Library Construction Protocol: | - |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | Forward |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina NextSeq 550 |
| Strand-Specific: | Specific |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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