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| Status |
Public on Nov 02, 2018 |
| Title |
twoN_CPC |
| Sample type |
SRA |
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| Source name |
CPC
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| Organism |
Mus musculus |
| Characteristics |
strain: FVB
age: Twelve-week-old
Sex: female
cell type: cardiac progenitor cells (CPCs)
ploidy: Diploid
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Adult c-Kit+ CPCs were isolated and expanded as previously described. Twelve-week-old FVB female mice were heparinized and anesthetized by ketamine-xylazine solution. Hearts were removed from the chest cavity and perfused on a Langendorff system at 1mL/min flow rate at 37ºC with perfusion buffer (NaCl, KCl, KH2PO4, Na2HPO4, MgSO4•7H2O, NaHCO3, KHCO3 HEPES, Taurine, Glucose, and BDM) for blood removal, and tissue was subsequently digested for 10-15 minutes with Liberase DH digestion buffer (Roche 05401089001, 5 mg/mL in perfusion buffer). Heart was collected in STOP buffer (10% Bovine calf serum, 12.5 µM CaCl2), minced into 1 mm3 and dissociated through pipetting. The non-myocyte population was separated through serial cell straining (100µm, 40µm, and 30µm pore size), followed by centrifugation at ~300 rfc for 10 minutes. Lin-cKit+ CPCs were obtained by immunomagnetic sorting with Lineage depletion kit and CD117-conjugated Microbeads (Miltenyi Biotech 130-048-102). Fresh isolated CPCs were separated for immediate single-cell RNA-Seq analysis or cultured in growth medium ((Dulbecco's modified Eagle's medium, 10% embryonic stem cells FBS, 1% insulin-transferrin-selenium, leukemia inhibitory factor [10 ng/ml], basic fibroblast growth factor [10 ng/ml], epidermal growth factor [20 ng/ml], L-glutamine [0.07mg/mL], 1% penicillin-streptomycin-glutamine, 0.1% gentamicin, 2% B27 supplement, 1% N2 supplement). Cells were plated on a pre-coated (0.1% porcine gelatin) cell culture-grade dish. Adherent cultures were expanded and passaged five times when 40% confluency was reached and used for single-cell RNA-seq.
The Chromium Single Cell 3' Library & Gel Bead Kit v2 (PN- 120237), Chromium Single Cell 3' Chip kit v2 (PN-120236) and Chromium i7 Multiplex Kit (PN-120262) were used according to the manufacturers instructions in the Chromium Single Cell 3' Reagents Kits V2 User Guide.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
The raw data was processed with the Cell Ranger pipeline (10X Genomics; version 2.0; 98 bp Read1, 8 bp i7 Index, and 26 bp Read2). Reads were aligned to the Mus musculus genome.
1036 diploid and 628 tetraploid freshly isolated CPC samples were recovered. Cells with less than 1000 genes or more than 10% of mitochondrial gene UMI count were filtered out and genes detected less than in three cells were filtered out. Altogether, 1664 cells and 15579 genes were kept for downstream analysis using Seurat R Package (v2.3.4).
Sequenced reads were aligned to the mouse reference genome mm10
Genome_build: mm10
Supplementary_files_format_and_content: values of digital expression matrix; rows are genes; columns are cells with cell barcode
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| Submission date |
Nov 01, 2018 |
| Last update date |
Nov 05, 2018 |
| Contact name |
Oscar E Echeagaray |
| E-mail(s) |
oscar.echeagaray@gmail.com
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| Organization name |
San Diego State University
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| Department |
Biology
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| Lab |
Sussman Lab
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| Street address |
5500 Campanile Drive
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| City |
San Diego |
| State/province |
CA |
| ZIP/Postal code |
92182 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE122057 |
Tetraploidy in Rodent Cardiac Stem Cells Confers Enhanced Biological Properties |
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| Relations |
| BioSample |
SAMN10358805 |
| SRA |
SRX4964906 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3453852_twoN_CPC.txt.gz |
15.9 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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