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: Three technical replicates for olfactory bulb and prefontal cortex tissues from Patient 1.
| Strategy: |
|
| Species: |
|
| Tissue: |
|
| Healthy Condition: |
|
| Growth Protocol: | - |
| Treatment Protocol: | - |
| Extract Protocol: | Nuclei were isolated from fresh frozen autopsy tissue following the 10x Genomics (Pleasanton, CA) recommended protocol using 0.1% Nonident NP40 lysis buffer. Suspensions were filtered through a 40 um mesh to remove debris and concentrated to a density of 1x10^6 nuclei/mL in Dulbecco's PBS + 0.04% BSA (Sigma Aldrich). Nuclei were processed according to Chromium 3’ Gene Expression V3 Kit (10X Genomics, Pleasanton, CA) using manufacturer’s guidelines. An estimated 15,000 nuclei were loaded onto each channel with a targeted cell recovery of 10,000 nuclei/sample. This was followed by sequencing on a S4 NovaSeq chip (Illumina Inc., San Diego, CA). Qubit 3 (Fisher Scientific) and 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) were used for quality check of cDNA. The output BAM file, a binary text file containing the sequence alignment data from sequencing, was processed through 10X Genomics Cell Ranger software v3.1.0. Sequencing generated scRNA-seq data with ~41,000 2x100bp reads per nucleus and 63,653 total cells sequenced |
| Library Construction Protocol: | - |
| Molecule Type: | polyA(+) RNA |
| Library Source: | |
| Library Layout: | PAIRED |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina NovaSeq 6000 |
| Strand-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 |
|---|