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| Status |
Public on Mar 15, 2022 |
| Title |
aMHC-Vegfb p7 WT |
| Sample type |
SRA |
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| Source name |
Cardiac endothelial cells
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| Organism |
Mus musculus |
| Characteristics |
strain background: C57Bl/6JCrl
age: postnatal day 7
genotype/variation: control wildtype (WT) animal
tissue: Cardiac endothelial cells
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| Treatment protocol |
For sample 3, we used an AAV-VEGF-B vector to transduce the VEGF-B expression in the heart. Sample 4 was injected with an empty AAV.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Mouse hearts were isolated for single-cell preparation as described previously. Hearts were harvested and coronary arteries were perfused briefly with buffer A (113 mM NaCl, 4.7 mM KCl, 0.6 mM KH2PO4, 0.6 mM Na2HPO4, 1.2 mM MgSO4, 12 mM NaHCO3, 10 mM KHCO3, 10 mM HEPES and 30 mM Taurine) to remove blood in a retrograde manner by cannulating the aorta before Collagenase type II (Worthington) was added to dissociate the ventricular cells. The dissociated cells were then allowed to sediment at 37°C for 10 minutes to allow cardiomyocytes to settle down as a pellet leaving behind a non-myocyte rich supernatant. The non-myocyte enriched supernatant was treated with rat anti-mouse CD16/CD32 (eBioscience, clone 2.4G2) at 4°C for 20 minutes before being incubated with fluorophore-conjugated antibodies at 4°C for 30 minutes. The following antibodies were used: anti-CD45-FITC (Biolegend, clone 30-F11), anti-PDGFR-PECy7 (eBioscience, clone APA5) and anti-CD105-PE (eBioscience, clone MJ7/18). Cell suspensions were triturated through a 40 µm Nylon cell strainer (Falcon) and then subjected to flow cytometry-based sorting using a Biorad S3e cell sorter.
Subsequent cDNA purification, amplification (12 cycles) and library construction (sample index PCR 14 cycles) was performed as instructed
Sample libraries were sequenced on the Illumina NovaSeq 6000 system using S1 flow cell (Illumina)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
aMHC-Vegfb p7 WT
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| Data processing |
The Cell Ranger v 2.1.1 mkfastq and count pipelines (10x Genomics, Pleasanton, CA) were used to demultiplex and convert Chromium single-cell 3’ RNA-sequencing barcodes and read data to FASTQ files and to generate aligned reads and gene-cell matrices.
Reads were aligned to mouse reference genome mm10
We used the Seurat R package (v2.3.4) for QC, filtering and analysis of the aggregated data (Butler et al. Nature Biotechnology 2018)
Cells were filtered based on the number of genes and the percentage of mitochondrial genes.
For further analysis the Pagoda2 package (version 0.0.0.9003) and Velocyto package and the velocyto.R package (version 0.6) have been used. Pseudotime analysis was done by using the monocle package (v2.8.0) with the default parameters.
Genome_build: mm10
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| Submission date |
Mar 19, 2019 |
| Last update date |
Mar 16, 2022 |
| Contact name |
Markus Räsänen |
| Organization name |
Wihuri Research Insitute
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| Street address |
Haartmaninkatu 8
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| City |
Helsinki |
| ZIP/Postal code |
00290 |
| Country |
Finland |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE128509 |
Single cell changes in postnatal P7 and in adult cardiac endothelium upon VEGF-B overexpression |
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| Relations |
| BioSample |
SAMN11164875 |
| SRA |
SRX5540726 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3678222_aMHC-Vegfb_WT_barcodes.tsv.gz |
12.1 Kb |
(ftp)(http) |
TSV |
| GSM3678222_aMHC-Vegfb_WT_genes.tsv.gz |
212.7 Kb |
(ftp)(http) |
TSV |
| GSM3678222_aMHC-Vegfb_WT_matrix.mtx.gz |
19.8 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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