Gene Expression
Nebulas
Gene Expression NebulasSummary: Despite effective treatment, HIV can persist in latent reservoirs, which represent a major obstacle towards HIV eradication. Targeting and reactivating latent cells is challenging due to the heterogeneous nature of HIV infected cells. Here, we used a primary model of HIV latency and single-cell RNA sequencing to characterize transcriptional heterogeneity during HIV latency and reactivation. Our analysis identified transcriptional programs leading to successful reactivation of HIV expression. We further validated our results using primary CD4+ T cells isolated from HIV+ individuals.
Overall Design: Human primary CD4+ T-cells were infected, cultured, and maintained in a resting, latent phenotype in order to generate a primary model of HIV latency. Latently infected cells were either left untreated, or exposed to SAHA or TCR stimulation, followed by single-cell isolation and single-cell RNA-seq (scRNA-Seq) analysis. Bulk RNA-Seq experiments were also performed as control. To validate the observed cellular heterogeneity in the primary model of HIV latency, we used primary CD4+ T cells isolated from HIV+ individuals. As for the primary HIV latency model, resting cells from HIV+ individuals were either not treated or TCR-treated before single cell isolation and single-cell RNA-Seq.
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| Growth Protocol: | For the latency model, CD4+ T cells were purified from the buffy coat of uninfected donors by Ficoll gradient separation followed by negative selection and magnetic separation using the human CD4+ T Cell enrichment kit (Stem Cell Technologies #17952). Then the cells were resuspended at 106 cells/ml in Activation Medium (Immunocult XF T cell expansion medium (Stem Cell Technologies #10981) supplemented with 100 U/ml IL-2 (RD #202-IL) and 25 l/ml of ImmunoCult_ Human CD3/CD28 T Cell Activator (Stem Cell Technologies #10971)). Three days post-TCR activation, cells (2 mio) were transduced with 1.2x105 TU of HIVGFP/VSV-G in presence of 5 g/ml polybrene by spinoculation (3h, 1500g, 25C). Cells were then washed with RPMI-1640 supplemented with 10% heat i-ctivated fetal calf serum (RNA10) and resuspended in 4 ml Expansion Medium (Immunocult XF T cell expansion medium supplemented with 100 U/ml IL-2). After three days, cells were washed, resuspended in RPMI without phenol red and sorted by FACS based on GFP expression. Infected GFP+ cells were resuspended in 0.5 ml Activation Medium and then maintained in Expansion Medium at 106 cells/ml for ~2.5 weeks. Cells were stimulated once again in Activation Medium and maintained in Expansion Medium for further 2.5 weeks. Cells were then allowed to revert to a resting phenotype by long-term culture in Latency Medium (50% RNA10/50% H80 feeder cell supeRNAtant supplemented with 40 U/ml IL-2) for 8 weeks. In parallel, blood was collected from two HIV-infected individuals participating in the Swiss HIV Cohort Study (http://www.shcs.ch), that were on antiretroviral therapy for more than 3 years, with undetectable viremia and a CD4 cell count above 300 for more than one year. Blood (~25 ml) was directly collected in 4 CPT tubes (BD Vacutainer CPT; BD Biosciences #362753) and processed for peripheral blood mononuclear cells (PBMC) isolation according to manufacturer__ instructions. Resting CD4+ T cells were purified by negative selection and magnetic separation using the human CD4+ T Cell enrichment kit supplemented with anti-HLA-DR, anti-CD25 and anti-CD69 (Stem Cell Technologies #19052/#17962), and resuspended in RNA10 (0.45 m filtered). |
| Treatment Protocol: | Human primary CD4+ T-cells were infected, cultured, and maintained in a resting, latent phenotype in order to generate a primary model of HIV latency. Latently infected cells were either left untreated; Human primary CD4+ T-cells were infected, cultured, and maintained in a resting, latent phenotype in order to generate a primary model of HIV latency. Latently infected cells were exposed to SAHA stimulation; Human primary CD4+ T-cells were infected, cultured, and maintained in a resting, latent phenotype in order to generate a primary model of HIV latency. Latently infected cells were exposed to TCR stimulation |
| Extract Protocol: | Cells were washed and collected for bulk or single-cell RNA-Seq. On one hand, cells (1 mio) were resuspended in 400 l lysis buffer of the ZRNADuet DNA/RNA miniprep (Zymo Research #D7001) and processed for RNA extraction according to manufacturer__ instructions, and used for bulk RNA-Seq (Illumina HiSeq Ribo-Zero TruSeq str RNA-Seq). On the other hand, cells were resuspended at 0.5-1x106 cells/ml in RNA10 (0.45 m filtered) and processed for single cell isolation and cDNA synthesis on a small 5-10 m fluidigm plate (fluidigm C1 Single Cell AutoPrep System, Clontech SMARTer Ultra Low RNA kit for Illumina sequencing), followed by single-cell RNA-Seq (Illumina Nextera XT DNA Sample Preparation). |
| Library Construction Protocol: | - |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | SINGLE |
| Library Strand: | -; Forward |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 2500 |
| Strand-Specific: | Unspecific; 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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