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| Status |
Public on Apr 01, 2024 |
| Title |
Choroid_mmu, scRNAseq |
| Sample type |
SRA |
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| Source name |
Choroid
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| Organism |
Mus musculus |
| Characteristics |
tissue: Choroid
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| Extracted molecule |
total RNA |
| Extraction protocol |
Mouse samples: Immune cells (CD45+ cells) were isolated from the choroid and pia mater of n=20 C57Bl/6J female mice (7 weeks of age) by FACS. Freshly dissected choroid and pia mater were pooled to obtain sufficient numbers of cells. The dissected tissues were transferred to 50 mL tubes containing dissection buffer (1x HBSS, no calcium, no magnesium, no phenol red; 5 mM glucose; 15 mM HEPES) and all processing was performed on ice. The tissues were passed through a 70 μm nylon cell strainer (Falcon 352350) using the plunger of a 5 mL syringe to obtain single cell suspensions, and then pelleted by centrifugation at 400 g for 5 min at 4 °C. Pia mater-enriched tissue was subsequently resuspended in 30% (v/v) Percoll (GE Healthcare 17089102) in 1 x PBS and centrifuged at 700 g for 10 min at 4 °C without a brake being applied. The myelinated (i.e., hydrophobic) layer formed at the top of the tube was discarded prior to resuspending the cell pellet. The number of viable cells was determined by exclusion of trypan blue stain as viewed by microscopy, assisted by use of a hemocytometer. Cells were subsequently resuspended in FACS buffer (3 mM EDTA, 0.1% (w/v) BSA, 1 x PBS, 100 μg/mL DNase I) and stained with fluorescent-conjugated antibodies (rat anti- mouse CD45-BV605, BD Biosciences 563890) for 30 min on ice, then centrifuged and resuspended in FACS buffer. Immune cells (CD45+) were isolated from stained cell suspensions using a BD FACS Aria IIIu (100 µm nozzle) at QIMR Berghofer Flow Cytometry Facility. Dead cells were identified and excluded from analysis by staining with propidium iodide (PI, 1 μg/ mL, BD Biosciences, Franklin Lakes, NJ, 556463). Human samples: Fresh (unfixed) human eye cups from a 45-year-old male donor with no history of eye disease or diabetes was obtained within 17 hours post-mortem from the Queensland Eye Bank. The vitreous and retinas were removed from the eye cups and choroid-RPE was subsequently dissected from the sclera. Choroid-RPE tissue was transferred into a 12-well plate and subjected to enzymatic digestion with collagenase type II (200 U/mL in HBSS (calcium, magnesium, no phenol red)) for 60 minutes at 37 °C, with tissue triturated every 15 minutes. After 60 minutes, choroid tissue was triturated with a pipette and then passed through a 70 µm cell strainer to generate a single cell suspension. The cell suspension was pelleted by centrifugation at 400 x g for 5 mins, resuspended in freezing media (DMEM/F12 containing 10% (v/v) DMSO and 10% (w/v) bovine serum albumin) and transferred to a Cool Cell freezing chamber at -80 °C for controlled freezing. Cells then were transferred to liquid nitrogen storage. For scRNA-seq studies, cells were thawed and pre-warmed DMEM/F12 containing 10% (w/v) BSA was added in a dropwise manner. The viability and yield of thawed cells were determined by trypan blue staining using a haemocytometer. Human choroidal immune cells were isolated from thawed cell suspensions by FACS. Briefly, thawed cell suspensions were incubated with 2.5 µg anti-human Fc block (BD Pharmingen 564220) for 15 mins, then centrifuged at 400 x g for 5 mins. The pellet was resuspended in FACS buffer containing BV421 Mouse Anti-Human CD45 antibody (BD Biosciences 563879) and incubated on ice for 30 mins. Cells were centrifuged at 400 x g for 5 mins then resuspended in FACS buffer for sorting. Live (propidium iodide negative) immune cells (CD45+) were sorted using a BD FACS Aria IIIu with a 100 µm nozzle.
scRNA-seq libraries were prepared using a Chromium single cell reaction 3’ v3.1 kit, following the manufacturer's instructions.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
10x Genomics
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| Data processing |
Demultiplexing, barcoding processing, gene counting and aggregation were completed using Cell Ranger software v7.0.0 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/using/tutorial_ov)
Downstream analysis (see below) was completed in R v4.0.5
QC, dimensionality reduction, clustering, and differential expression analysis was completed using the R package Seurat (https://satijalab.org/seurat/)
Functional annotation of GE genes to KEGG pathways and GO terms was completed using the R package clusterProfiler (https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html)
Assembly: mm10, hg38
Supplementary files format and content: Quality-filtered barcodes (cell IDs), features (genes) and count matrix, generated by Cell Ranger.
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| Submission date |
Jan 16, 2024 |
| Last update date |
Apr 01, 2024 |
| Contact name |
Paul James Whatmore |
| E-mail(s) |
pauljwhatmore@gmail.com
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| Organization name |
Queensland University of Technology
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| Department |
eResearch
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| Street address |
2 George St
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| City |
Brisbane |
| State/province |
Queensland |
| ZIP/Postal code |
4000 |
| Country |
Australia |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE253419 |
Tissue-specific immune transcriptional signatures in the bordering tissues of the mouse brain and retina |
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| Relations |
| BioSample |
SAMN39463641 |
| SRA |
SRX23254040 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8020423_Choroid_mmu_barcodes.tsv.gz |
11.6 Kb |
(ftp)(http) |
TSV |
| GSM8020423_Choroid_mmu_features.tsv.gz |
284.1 Kb |
(ftp)(http) |
TSV |
| GSM8020423_Choroid_mmu_matrix.mtx.gz |
15.8 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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