Gene Expression
Nebulas
Gene Expression NebulasSummary: Our work illustrates how high-resolution molecular and spatial profiling of COVID-19 patient tissues collected during rapid autopsies can serve as a hypothesisgenerating tool to identify key mediators driving the pathophysiology of COVID-19 for diagnostic and therapeutic target testing. Here we employ bulk RNA sequencing to identify key regulators of COVID-19 and list specific mediators for further study as potential diagnostic and therapeutic targets. We use single-nuclei RNA sequencing to highlight the diversity and heterogeneity of coronavirus receptors within the brain, suggesting that it will be critical to expand the focus from ACE2 to include other receptors, such as BSG and ANPEP, and we perform digital spatial profiling of lung and lymph node tissue to compare two patients with different clinical courses and symptomatology.
Overall Design: Two technical replicates across ten tissues (Patient 1) with two technical replicates of a pool of brain RNA from multiple donors as control (no SARS-CoV-2 infection).
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| Growth Protocol: | - |
| Treatment Protocol: | - |
| Extract Protocol: | Total RNA was extracted from the autopsy tissues by first flash freezing each tissue in liquid nitrogen and then using the CryoPREP Tissue Disruption System (Covaris) to pulverize the tissue into a fine powder. The powder was then immediately resuspended in cell lysis buffer and RNA was extracted using the ReliaPrep RNA Tissue Miniprep System (Promega, #Z6112) by following the manufacturer’s instructions. A DNase digestion was also performed during the RNA extraction workflow to remove any potential contamination from carryover genomic DNA (reagents included in the kit). The extracted RNA was then purified using 1.8X RNAClean XP beads (Beckman Coulter) to remove any molecular impurities. |
| Library Construction Protocol: | Stranded total RNA libraries were generated according to the manufacturer's instructions, using the TruSeq Stranded Total RNA Gold kit for Illumina sequencing |
| Molecule Type: | rRNA- RNA |
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| Library Layout: | PAIRED |
| Library Strand: | Forward |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina NovaSeq 6000 |
| 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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