Gene Expression
Nebulas
Gene Expression NebulasSummary: Angiotensin-Converting Enzyme 2 (ACE2) binds SARS-CoV-2 spike protein and facilitates viral entry into the cell. ACE2 is expressed in the kidney and renal injury strongly impacts COVID-19 prognosis. In addition, a short, interferon-inducible isoform of ACE2, dACE2 was recently identified. Change in ACE2 expression has already been linked to several human nephropathies. However, these changes were not analyzed in context of dACE2 and regulation of ACE2 expression in the kidney remains unclear. Human primary Renal Proximal Tubule cells were used for all experiments. ChIP-seq analysis of histone modifications (H3K27ac, H3K4me3, K3K4me1), RNA polymerase loading and DNAse hypersensitive sites (DHS) were used to identify gene regulatory elements in the ACE2 locus. qRT-PCR was used to assess mRNA expression of ACE2, dACE2, TMPRSS2 and STAT1. RNA-seq was used to identify changes in global gene expression after cytokine treatment. Putative regulatory elements controlling dACE2 expression were identified using ChIP-seq profiles and RNA-seq mapping. qRT-PCR differentiating between ACE2 and dACE2 revealed that only dACE2 is interferon inducible. dACE2 mRNA was upregulated 300- and 600-fold by IFNα and IFNβ, respectively, while full length ACE2 was unchanged. Interferon stimulation also resulted in elevated STAT1 expression. JAK inhibitor ruxolitinib ablated both STAT1 and dACE2 expression after interferon treatment. RNA-seq analysis revealed approximately 1200 genes induced by IFNβ, largely innate immune response-related. We showed that human primary proximal tubules express ACE2 and its interferon-inducible short isoform, dACE2, from an intronic promoter. We also deliver new datasets identifying cytokine response genes in proximal tubule cells.
Overall Design: RNA-seq was used to characterize ACE2 locus and gene expression profiles in cytokine stimulation
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| Growth Protocol: | Primary Human Renal Proximal Tubule Epithelial Cells (ATCC® PCS-400-010™) were cultured in low-serum medium consisting of Renal Epithelial Cell Basal Medium (ATCC® PCS-400-030™) with Renal Epithelial Cell Growth Kit (ATCC® PCS-400-040™), Penicillin-Streptomycin-Amphotericin B Solution (ATCC® PCS-999-002™) and Phenol Red (ATCC® PCS-999-001™) added. |
| Treatment Protocol: | human primary proximal tubule (HPPT) cells were stimulated with IFNα, IFNβ, IFNγ, TNFα, IL-6 and IL-1β for 12 hours in concentration of 10 ng/ml |
| Extract Protocol: | Total RNA was isolated from the cytokine-stimulated cells using PureLink™ RNA Mini Kit (Invitrogen) and libraries for sequencing were prepared according to the manufacturer’s instructions with TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold (Illumina, RS-122-2301). |
| Library Construction Protocol: | Libraries for sequencing were prepared using standard Illumina protocols. |
| Molecule Type: | rRNA- RNA |
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| Library Layout: | SINGLE |
| Library Strand: | Forward; - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 3000 |
| Strand-Specific: | Specific; Unspecific |
| 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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