Gene Expression
Nebulas
Gene Expression NebulasSummary: Purpose: To better characterise the population structure of primary bovine retinal microvascular endothelial cells (RMECs) based upon their individual transcriptomes. Methods: Individual RMECs were captured on the Fluidigm C1 system (Fluidigm), cDNA libraries were prepared using a Nextera XT kit and sequencing performed on a NextSeq (Illumina). Results: Application of a single cell RNA-seq analysis workflow showed that RMECs form a relatively homogeneous population in culture, with the main subgroup being proliferating cells. Expression of markers from along the arteriovenous tree suggests that most cells originate from capillaries. An in silico model of the blood retinal barrier was created, including junctional proteins not previously reported within the retinal vasculature. Numerous alternative splicing events involving exons within microvascular barrier genes were observed and in many cases individual cells expressed exclusively one isoform. Conclusions: We have optimised a workflow for single-cell transcriptomics in primary RMECs. Our results have provided fundamental insights into the genes involved in retinal microvascular barrier formation
Overall Design: Single cell RNA-seq analysis of retinal microvascular endothelial cells from two separate Fluidigm C1 runs, 49 cells from run 1 and 51 cells from run 2.
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| Extract Protocol: | RMECs were isolated from bovine retinas according to (Stitt, Chakravarthy, Archer, & Gardiner, 1995). RMECs were maintained in DMEM supplemented with 0.1mg/ml Primocin, 0.38渭g/ml insulin, 1mg/ml heparin and 10% (v/v) porcine serum (PS). BRECs were passaged at approximately 80% confluency in a 1:3 ratio and grown until confluency at passage 3 for Fluidigm C1 runs . RMECs were dissociated form flask using TryPLE Express dissociation reagent and single cells were captured on Fluidigm C1 Microfluidics chip. |
| Library Construction Protocol: | - |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | PAIRED |
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| Platform: | ILLUMINA |
| Instrument Model: | Illumina NextSeq 500 |
| 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 |
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