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| Status |
Public on Aug 18, 2020 |
| Title |
G1 |
| Sample type |
SRA |
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| Source name |
day 5
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL6/J
Sex: Male
condition: Myocardial infarction
cell type: CD45+ total leukocytes from heart
time point: Day 5
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| Treatment protocol |
Mice were anesthetized with isoflurane (4.0%), received an intravenous injection of 5µg of anti-CD45.2-APC (clone 104, ThermoFisher Scientific) in 100µl of PBS, and left under isoflurane anesthesia for 5-10 minutes before being killed by cervical dislocation. Induction of myocardial infarction was macroscopically confirmed and mice received an intracardiac perfusion of PBS and the hearts were collected. The right ventricle was removed as well as the viable myocardium right above the ligature site, so that the infarcted area, its border zone and the adjacent viable myocardium were processed. A similar part of the left ventricle was processed for non-infarcted hearts. The excised hearts were rinsed in 4°C RPMI and gently massaged to expel excess blood, minced with surgery scissors and digested in RPMI containing 450U/ml collagenase I (Sigma-Aldrich C0130), 125U/ml collagenase XI (Sigma-Aldrich C7657), 60U/ml Hyaluronidase (Sigma-Aldrich H3506) for 1 hour at 37°C in a Thermomixer (Eppendorf) with shaking set at 1000rpm. The resulting cell suspension was filtered through a 70µm cell strainer, and incompletely digested pieces of myocardium were gently dissociated using a syringe plunger. Cells were then washed in 50ml 4°C PBS containing 1% Fetal Bovine Serum (centrifugation 8 minutes at 300rcf).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cardiac cells were collected at different time points after myocardial infarction (no infarct/Day 0: n=5; Day 1: n=5; Day 3: n=5, Day 5: n=5; Day 7: n=3) as described above. Cell suspensions were incubated for 10 minutes at 4°C with 10μg/ml Fc Block (purified rat anti-mouse CD16/CD32, Biolegend TruStain FcX anti-mouse) to block non-specific binding of antibodies to Fc Receptors and then incubated at 4°C for 30 minutes with anti-CD45-PE (1:300, Thermofisher Scientific, Clone 30-F11), anti-Ly6G-V450 (1:300, BD Biosciences, Clone 1A8) and Fixable Viability Staining e780 (1:1000, Thermofisher Scientific). All staining and washing steps were performed in PBS containing 1% FCS, except the last wash and sorting that were made in PBS supplemented with 0.04% BSA (Sigma Aldrich, SRE0036). Right before sorting, cells were passed through a 40µm cell strainer. In this experiment, a neutrophil reduction step was performed to increase the proportion of monocyte/macrophages at early time points. Viable CD45.2 (intravenous)-negative/CD45+ cells were sorted using a FACS-Aria III (BD Biosciences) with a 100µm nozzle, counted and loaded into the 10X Genomics Chromium.
Cells were loaded in the 10X Genomics Chromium at concentrations recommended by the manufacturer. Libraries were prepared with Chromium Single Cell 3’ Reagents Kit v2 Chemistry.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Data were analyzed using Cell Ranger™ v3.0.1 pipelines which is available in 10x Genomics website.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: h5: Contains data corresponding to the observed molecules, as well as data about the libraries, feature set(s), and barcode lists used for the analysis.
Supplementary_files_format_and_content: *tsv, *mtx: Gene barcode matrices and feature barcode matrix.
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| Submission date |
Aug 02, 2019 |
| Last update date |
Jun 17, 2022 |
| Contact name |
Antoine-Emmanuel Saliba |
| E-mail(s) |
emmanuel.saliba@helmholtz-hzi.de
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| Phone |
+49-931-31-81341
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| Organization name |
Helmholtz Institute for RNA-based Infection Research
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| Street address |
Josef-Schneider-Straße 2 / D15
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| City |
Würzburg |
| ZIP/Postal code |
97080 |
| Country |
Germany |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE135310 |
Time series single cell transcriptomics of cardiac inflammation after myocardial infarction in mice |
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| Relations |
| BioSample |
SAMN12441298 |
| SRA |
SRX6647051 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4005127_G1_mm10_barcodes.tsv.gz |
4.9 Kb |
(ftp)(http) |
TSV |
| GSM4005127_G1_mm10_genes.tsv.gz |
212.7 Kb |
(ftp)(http) |
TSV |
| GSM4005127_G1_mm10_matrix.mtx.gz |
6.2 Mb |
(ftp)(http) |
MTX |
| GSM4005127_G1_molecule_info.h5 |
139.9 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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