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| Status |
Public on May 29, 2024 |
| Title |
Grafted vein, canine 1, snRNA-seq |
| Sample type |
SRA |
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| Source name |
Cephalic vein
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| Organism |
Canis lupus familiaris |
| Characteristics |
tissue: Cephalic vein
cell type: Endothelial cells, smooth muscle cells, fibroblasts, myeloid cells
Sex: Male
treatment: Grafted
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| Treatment protocol |
Bilateral upper limb cephalic vein harvest was performed on 25 kg male (n=3) or female (n=1) mongrel dogs (Marshall Bioresources). General anesthesia was established and maintained with an initial sodium pentothal injection and subsequent 1% isofluorane inhalation after orotracheal intubation. Vein harvest was performed in conventional fashion. The cephalic vein was identified and exposed along the anterior surface from the wrist to the upper forearm. The distal portion of the vein was then ligated and transected. The end of the transected vein was cannulated with a metal cannula and secured with a silk suture. The vein was then sequentially distended by injecting a saline solution (0.9% sodium chloride, papaverine hydrochloride 60 mg, and 43 mg of bivalirudin) while occluding the outflow. Intervening tributaries were identified and ligated with silk sutures. A total length of 15 cm of vein was harvested. Total vein distension and harvest time was approximately 30 minutes. The cephalic vein following distention, along with a separate segment of the undistended cephalic vein, which served as the experimental control, were excised. Samples were collected and immediately snap-frozen in cryovials using liquid nitrogen for single-nuclei analysis or embedded in OCT blocks and flash-frozen in an isopentane and liquid nitrogen bath for spatial transcriptomics analysis. Systemic bivalirudin was then infused before the creation of the anastomoses. Bilateral end-to-side anastomoses were created on the vein with 7-0 Prolene sutures. The intervening segment of the carotid artery was then ligated, creating preferential flow through the graft. After hemostasis was achieved, the wounds were closed in three layers with 3-0 vicryl sutures. The patency of the graft was confirmed through direct palpation and ultrasound imaging. At 24 hours, harvest was performed with the dog under general anesthesia. Vein graft samples were then collected and processed as previously described for cryopreservation and OCT embedding.
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| Extracted molecule |
nuclear RNA |
| Extraction protocol |
For single-nuclei analysis, we used the sample prep user guide (10x Genomics, CG000505, Rev A) for the Chromium Nuclei Isolation Kit with RNase Inhibitor (10x Genomics, 1000494) to isolate nuclei from flash-frozen tissues. The various buffers (lysis, debris removal, wash, and resuspension buffers) were prepared using kit components according to user guide instructions (10x Genomics, CG000505, Rev A). Briefly, ~50 mg of tissue, cut into smaller pieces to enable better lysis of cells, was added to pre-chilled sample dissociation tubes and dissociated by adding lysis buffer followed by multiple strokes with the pestle against the dissociation tubes. Following incubation on ice for 10 minutes, the dissociated tissue was added to the nuclei isolation column, placed in a pre-chilled collection tube, and spun at 16,000 g for 20 sec at 4oC. The flowthrough was collected, vortexed to resuspend the nuclei, and spun at 500g, 3 min, 4oC. The pellet was then resuspended in debris removal buffer, pipet mixed, and spun at 700 g, 10 min, 4oC. After discarding the supernatant, the pellet was washed with wash and resuspension buffer by spinning at 500 g, 5min, 4oC. For counting, the nuclei were stained with acridine orange/propidium iodide stain (Logos Biosystems) and counted using a LUNA-FX7 cell counter (Logos Biosystems). The nuclei pellet was resuspended in wash and resuspension buffer to get the desired nuclei concentration (700-1200 nuclei/ml).
For snRNA-seq, gene expression libraries were prepared immediately from the isolated nuclei with the Chromium Next GEM Single Cell 3’ v3.1 reagent kits (10x Genomics, 1000268). Briefly, GEMs were prepared using the 10x Genomics controller followed by RT-PCR and cDNA amplification. The cDNA was then used to prepare snRNA-seq libraries according to the user guide (10x Genomics, CG000315 rev. B). The quality of the cDNA and final library preps was assessed using DNA HS bioanalyzer kits (Agilent), and concentration was estimated using a Qubit fluorometer (Thermo Fisher Scientific).
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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 |
C1-SN3, 10x Genomics, Chromium Nuclei
GVN2_C1-SN3
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| Data processing |
For snRNA-seq samples, demultiplexing, barcoded processing, gene counting, and aggregation were performed using the 10x CellRanger software (v.7.0.0).
Assembly: Dog10K_Boxer_Tasha_1.0
Supplementary files format and content: Tab-separated value files for barcodes and genes with matrix files
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| Submission date |
Apr 05, 2024 |
| Last update date |
May 29, 2024 |
| Contact name |
Marina Michaud |
| E-mail(s) |
marina.michaud@emory.edu
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| Phone |
4043078082
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| Organization name |
Emory University
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| Department |
School of Medicine
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| Lab |
Bhasin Lab
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| Street address |
28 Cass Station Dr NW
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| City |
Cartersville |
| State/province |
GA |
| ZIP/Postal code |
30120 |
| Country |
USA |
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| Platform ID |
GPL25760 |
| Series (1) |
| GSE263280 |
Single-nuclei expression of canine veins during carotid-cartoid vein bypass implantation |
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| Relations |
| BioSample |
SAMN40759500 |
| SRA |
SRX24164705 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8189147_C1-SN3_barcodes.tsv.gz |
14.5 Kb |
(ftp)(http) |
TSV |
| GSM8189147_C1-SN3_features.tsv.gz |
300.3 Kb |
(ftp)(http) |
TSV |
| GSM8189147_C1-SN3_matrix.mtx.gz |
17.9 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
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