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| Status |
Public on Apr 01, 2021 |
| Title |
Single nucleus human perivascuar adipose tissue 3 |
| Sample type |
SRA |
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| Source name |
PVAT
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| Organism |
Homo sapiens |
| Characteristics |
age: 61 years
gender: Male
tissue: PVAT
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| Extracted molecule |
total RNA |
| Extraction protocol |
For single cell, Aorta were isolated from mice, minced and placed into digestion medium (DMEM, Collagenase D: 6.1mg/ml (Roche), Dispase II: 2.4 mg/ml (Roche) and placed at 37oC with constant agitation at 200 rpm. To enrich for cell populations with differential sensitivity to digestion, we utilized the following procedures. For embryonic/newborn mice, 20% of the digestion was quenched at 15 minutes, 20% at 20 minutes and the remaining 60% at 25 minutes (embryos/perinatal). For adult aortas 50% of the digestion was quenched at 30 minutes, 50% at 60 minutes. Single cells were isolated from dissected aortas and flow sorted to isolate live (DAPI-) cells and remove debris. Cells were collected as either CD45+ (immune) and CD45- (non-immune) cells. For adult single cell RNA Sequencing, CD45+ cells were mixed with CD45- cells to achieve a proportion of ~20%. For single nuclei experiments, 40-80 mg of frozen human PVAT was dounce homogenized using a glass homogenizer (Sigma-Aldrich, 7 mL) on ice in 2.5 mL homogenization buffer (0.32 M sucrose (Sigma, RNase & DNase free ultra pure grade), 5 mM CaCl2 (Sigma), 3 mM MgAc2 (Sigma-Aldrich), 10 mM Tris-HCl pH 8.0 (FisherChem), 0.1% TritonX-100 (Amresco), 0.1 mM EDTA (Fischer), protease inhibitor cocktail (Roche)) and layered into ultracentrifuge tubes (Beckman Coulter) over 12 mL sucrose cushion (1.8 M sucrose, 10 mM Tris-HCl pH 8.0 (FisherChem), 3 mM MgAc2 (Sigma-Aldrich), protease inhibitor cocktail (Roche) and topped with 14.5mL homogenization buffer. Samples were ultracentrifuged (Beckman Coulter L-70K) using a swinging bucket rotor (Beckman Coulter SW28) at 25,000 rpm for 2 hrs at 4°C. The supernatant was aspirated to remove debris and the nuclear pellet was resuspended in 1 mL of DPBS (Corning) with RNase inhibitor (Lucigen). Samples were filtered and nuclei were counted before loading for snRNA-Seq
For single cell experiments, single cell transcriptomes were collected previously using the 10X Genomics platform with v2 (perinatal) and v3 (adult) chemistry (10× Genomics, Pleasanton, CA) and sequenced on Illumina HiSeq 4000. For single nucleus experiments, single nucleus transcriptomes were collected using the 10X Genomics platform v3 (adult) chemistry (10× Genomics, Pleasanton, CA) and sequenced on Illumina NovaSeq 6000.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
10X Genomics Single Nucleus RNA-Seq
hPVAT.Integrated_annotated_merged.RDS
hPVAT.Integrated_raw.rds
hPVAT.Integrated_annotated.rds
hPVAT.Subclustered_annotated.RDS
hPVAT.Subclustered_raw.RDS
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| Data processing |
CellRanger v3
Seurat v3
Genome_build: mm10; GRCh38
Supplementary_files_format_and_content: CellRanger Outputs (Barcodes.tsv, genes.tsv, matrix.mtx) and Seuratv3 RDS Files,
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| Submission date |
Feb 08, 2021 |
| Last update date |
Apr 02, 2021 |
| Contact name |
Patrick Seale |
| E-mail(s) |
sealep@pennmedicine.upenn.edu
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| Organization name |
University of Pennsylvania
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| Street address |
Smilow Center for Translational Research, 12th Floor 3400 Civic Center Blvd.
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE164528 |
Defining the lineage of thermogenic perivascular adipose tissue |
| GSE166355 |
Defining the lineage of thermogenic perivascular adipose tissue [Thoracic PVAT] |
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| Relations |
| BioSample |
SAMN17833939 |
| SRA |
SRX10048757 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5068998_hPVAT3_barcodes.tsv.gz |
27.8 Kb |
(ftp)(http) |
TSV |
| GSM5068998_hPVAT3_genes.tsv.gz |
287.6 Kb |
(ftp)(http) |
TSV |
| GSM5068998_hPVAT3_matrix.mtx.gz |
39.2 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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