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| Status |
Public on Nov 04, 2019 |
| Title |
AT-21-VAT-CD34- |
| Sample type |
SRA |
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| Source name |
adipose derived stromal vascular fraction (SVF)
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| Organism |
Homo sapiens |
| Characteristics |
subject status: obese individual
diabetic status: Non Diabetic
tissue: Visceral adipose tissue
cd34 sorting: CD34_Negative
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Adipose tissue was digested with collagenase according to a modification of the Robdell method (Tchernof et al., 2006). Briefly, adipose tissue samples were digested with collagenase type 1 in Krebs-Ringer-Henseleit (KRH) buffer for 45 minutes at 37°C. Cell suspensions containing mature adipocytes and the stromal-vascular fraction (SVF) were then filtered with a nylon mesh and washed 3 times with KRH buffer. The nature of the buoyancy adipocytes allows them to float to the surface. Mature adipocytes were discarded and the remaining solution containing the SVF was centrifuged 1500 rpm for 5 minutes. The pellet was washed with pre-adipocyte growth medium (PGM) (DMEM-F12 supplemented with 10% calf serum, 1% penicillin-streptomycin, 17µM pantothenic acid, 33µM biotin, 100µM ascorbic acid and 2,5 µm/ml amphoB) followed by a second centrifugation. Cells were cryopreserved using freezing medium (PGM supplemented with 40% FBS and 10% DMSO). The medium was added to the pellet and was frozen with a temperature gradient (-1 °C/ minutes) and store at -80°C until analysis. The FACS sorted SVF samples were incubated with primary antibody (CD34) and sorted using FACSAria II
Cells from the SVF were thawed and serially diluted in DMEM/F12 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in 15ml conical tube to a volume of 14ml. They were centrifuged at 300 rcf for 10 minutes and the supernatant was removed and discarded. The pelleted cells were suspended in an adequate amount of DMEM/F12 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin and transferred to a 1.5ml microfuge tube for counting. 10 µl of the suspension was used for a cell count and viability assay on a Countess II Automated Cell Counter (Invitrogen) to determine the volume of suspension to be used in the 10x Chromium Single Cell Library protocol. The desired number of viable cells to be loaded onto the Chromium Single Cell A Chip (10x Genomics) was 12,000 cells. After cell capture, the remaining cells were centrifuged at 300 rcf for 10 minutes and cryopreserved using freezing medium (Recovery Cell Culture Freezing Medium, Thermo Fisher Scientific). The cells were suspended in freezing medium and frozen with a temperature gradient of -1⁰C per minute. Cell capture, cDNA amplification, and library preparation were performed using the Chromium Single Cell 3' Library & Gel Bead Kit v2 (10x Genomics), according to the manufacturer’s protocol. The final libraries were analyzed using a TapeStation to determine library size and a Qubit Broad Range dsDNA assay to determine library concentration and sequenced using Illumina HiSeq.
Chromium Single Cell 3’ Library
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
AT-E21_IA_Neg
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| Data processing |
Primary Analysis of demultiplexing, barcode correcting, alignment and generation of gene expression matrix was done using cellranger v.2.1.0 from 10X Genomics using GRCh38 as reference
Seurat v. 2.3.3 was then used for downstream analysis of filtering, Normalization, clustering and dimensionality reduction.
All Samples were merged together and we removed cells with low gene (<200), low unique molecular identifier (UMI, < 200) and high mitochondrial gene expression (> 20%). In order to exclude doublets, we also removed cells with high Gene (>2,500) and high UMI (>13,750).
Clusters obtained from Seurat was annotated using SingleR
genome build: GRCh38
processed data files format and content: SVF_Normalised_Data.txt: Tab delimited file containing normalised, log transformed expression matrix.
processed data files format and content: Discovery_Cohort_barcodes.tsv: Barcode file of filtered feature barcode matrix obtained from Cell Ranger aggregate results of discovery cohort.
processed data files format and content: Discovery_Cohort_CellAnnotation.txt: Cell Annotation for the discovery cohort.
processed data files format and content: Discovery_Cohort_genes.tsv: Genes file of filtered feature barcode matrix obtained from Cell Ranger aggregate results of discovery cohort.
processed data files format and content: Discovery_Cohort_matrix.mtx: Matrix file of filtered feature barcode matrix obtained from Cell Ranger aggregate results of discovery cohort.
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| Submission date |
Apr 04, 2019 |
| Last update date |
Jan 08, 2020 |
| Contact name |
Elin Grundberg |
| Organization name |
CHILDREN'S MERCY HOSP (KANSAS CITY, MO)
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| Street address |
2401 Gillham Road
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| City |
Kansas City |
| State/province |
Missouri |
| ZIP/Postal code |
64108 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE129363 |
Single Cell RNASeq profiling of stromal vascular fraction from Subcutaneous and visceral adipose tissue |
| GSE136230 |
Single-cell analysis of human adipose tissue |
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| Relations |
| BioSample |
SAMN11347935 |
| SRA |
SRX5644477 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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