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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jan 27, 2023 |
| Title |
Cardiovascular progenitor cells (FACS d4.5 RA+ eGFP+) |
| Sample type |
SRA |
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| Source name |
Cardiovascular progenitor cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: in vitro differentiated human embryonic stem cells (hESC)
pluripotent stem cell line: a double reporter hESC-03 line expressing eGFP and mCherry under the control of the endogenous NKX2-5 and TBX5 locus respectively
treatment: version of protocol: 0.5 uM RA, 4 days
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| Treatment protocol |
Two versions of protocol were applied: without RA (RA-) between 40h (d1.5) and d5.5 or with 0.5 µM RA for 4 days between 40h (d1.5) and d5.5 (RA+)
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| Growth protocol |
Human embryonic stem cells were differentiated as described in methods of Zawada et al., (2021) using a stepwise growth-factor based protocol to direct hESCs towards CMs through defined developmental stages of primitive-like cells and early cardiovascular progenitor cells.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
For scRNASeq at d1.5, cells were washed two times with PBS, dissociated with Accutase (3-5 min, 37°C), centrifuged at 1200 rpm for 5 min, resuspended in 0.04% BSA in PBS, filtered through a 40 μm filter and counted. For scRNASeq at d4.5 of differentiation, cells were subjected to sorting procedure as described in methods of Zawada et al. (2021). Cells were sorted based on mCherry and eGFP expression and collected into 10% FCS in PBS. Then cells were centrifuged, washed in 0.04% BSA in PBS, centrifuged again, filtered through a 40 μm filter and resuspended in 0.04% BSA in PBS for counting. For scRNA-Seq at d30 of differentiation of aggregates, aggregates were dissociated with papain as described in methods of Zawada et al. (2021), filtered through 40 μm filter, centrifuged at 1000 rpm for 3 min and resuspended in 0.04% BSA in PBS for counting. After counting, 10 000 cells for each sample were processed using Chromium Single Cell 3’ Library & Gel Bead Kit v3.1 (1000075, 10x Genomics) Chromium Single Cell B Chip Kit (1000073, 10x Genomics) and Chromium i7 Multiplex Kit (220103, 10x Genomics) to generate Gel Bead-In-EMulsions (GEMs) and single cell sequencing libraries.
10 000 cells per sample were processed using Chromium Single Cell 3’ Library & Gel Bead Kit v3.1 (1000075, 10x Genomics) Chromium Single Cell B Chip Kit (1000073, 10x Genomics) and Chromium i7 Multiplex Kit (220103, 10x Genomics) to generate Gel Bead-In-EMulsions (GEMs) and single cell sequencing libraries. Libraries were pooled and sequenced using the NextSeq 500/500 (Ilumina; High Output v2 kit 75 cycles v2.5 flow cell) with 28 cycles in read1 for the 10x barcodes and UMIs in and 8 cycles i7 index read and 58 cycles for cDNA in read2 with a read depth of at least 20 000 pair reads per cell.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Sample2
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| Data processing |
Cell Ranger workflow using the DropletUtils was used to generate FASTQ and bam files
Genome_build: hg19
Supplementary_files_format_and_content: Cell Ranger output
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| Submission date |
Mar 01, 2022 |
| Last update date |
Jan 27, 2023 |
| Contact name |
Gianluca Santamaria |
| E-mail(s) |
santamariagianluca@tum.de
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| Organization name |
Klinikum rechts der Isar – Technical University Munich
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| Department |
Department of Medicine I
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| Lab |
Molecular Cardiology
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| Street address |
Ismaninger Strasse 22
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| City |
Munich |
| State/province |
Germany |
| ZIP/Postal code |
81675 |
| Country |
Germany |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE197660 |
Retinoic acid signaling modulation guides in vitro specification of human heart field-specific progenitor pools |
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| Relations |
| BioSample |
SAMN26342506 |
| SRA |
SRX14330396 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5924610_Sample2_processed_data.tar.gz |
66.2 Mb |
(ftp)(http) |
TAR |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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