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| Status |
Public on Dec 03, 2024 |
| Title |
ROBO_CD31 |
| Sample type |
SRA |
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| Source name |
Retina
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| Organism |
Mus musculus |
| Characteristics |
tissue: Retina
cell type: Edothelial
treatment: ROBO1/2 blocking antibodies
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| Treatment protocol |
For antibody treatment, 10mg/kg of antibodies or isotype control IgG were diluted in 200uL of sterile 0.9% NaCl and given i.p. at P12, P14 and P16. For IPI-549 treatment, 15mg/kg of the inhibitor was diluted in 200uL of sterile vehicle (0.9% NaCl 2%DMSO) and given i.p. daily from P12 to P16; and control mice were treated i.p. with 200uL of vehicle.
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| Growth protocol |
For the induction of OIR, the mother and P7 pups were placed in 75% O2 until P12. Upon return to room air, the pups were placed for adoption with a nursing mother and treated. Eyes were collected at P17.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Briefly, 8 retinas per group were dissected and dissociated with the Neural Tissue Dissociation kit (P) (Miltenyi Biotec, Cat#130- 092-628). The digestion was stopped by adding BSA (5% final) and the cells were immediately filtered through a 70 mm cell strainer. After centrifugation (600g for 5 min), cells were resuspended in PBS supplemented with 2mM EDTA and 0.5% BSA. The single cell suspension was enriched for ECs and immune cells using CD31 and CD45 MicroBeads (Miltenyi Biotec) according to the manufacturer’s instructions.
Single cell suspensions of retinal cells were resuspended in PBS containing 0.04% ultra-pure BSA. scRNA-seq libraries were prepared using the Chromium Single Cell 30 Reagent Kits v3.1 (10x Genomics; Pleasanton, CA, USA) according to the manufacturer’s instructions. The target cell recovery for each library was 10,000. Generated libraries were sequenced on an Illumina HiSeq4000.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Demultiplexing, barcode processing and gene quantififcation were performed with CellRanger v2.1.1
After generation of gene expression matrices using CellRanger (10x Genomics), processing and analysis of the data were performed using the Seurat R-package (v4)
First, quality control steps filtered out genes that were expressed in less than 10 cells and cells that expressed fewer than 100 genes. Lastly, only cells which had fewer than 60000 counts, between 500 and 7500 genes and less than 20% of the unique molecular identifiers (UMIs) originating from mitochondrial genes were retained for further analysis.
For clustering and visualization, individual samples were normalized using the SCTransform function. Seurat SCTransform dataset integration was performed to merge P17 Control and Anti-Robo1/2 datasets to correct for batch effects. Dimensional reduction was performed using Uniform Manifold Approximation and Projection (UMAP) as implemented in the RunUMAP function, where 15 dimensions were used for the dims parameter and all other settings were default
Assembly: mm10
Supplementary files format and content: h5Seurat files with counts and scaled data
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| Submission date |
Oct 18, 2024 |
| Last update date |
Dec 03, 2024 |
| Contact name |
Anne Eichmann |
| Organization name |
Yale University
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| Department |
Cardiovascular research center
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| Lab |
Eichmann lab
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| Street address |
300 George St
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| City |
New Haven |
| State/province |
CT |
| ZIP/Postal code |
06511 |
| Country |
USA |
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| Platform ID |
GPL21103 |
| Series (1) |
| GSE279825 |
Monoclonal antibodies that block Roundabout 1 and 2 signaling target pathological ocular neovascularization through myeloid cells |
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| Relations |
| BioSample |
SAMN44346393 |
| SRA |
SRX26457815 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8581623_ROBO_CD31_MMT_barcodes.tsv.gz |
4.3 Mb |
(ftp)(http) |
TSV |
| GSM8581623_ROBO_CD31_MMT_matrix.mtx.gz |
123.1 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
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