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| Status |
Public on Aug 18, 2020 |
| Title |
H10 |
| Sample type |
SRA |
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| Source name |
CITEseq/Multiplexed Day1-3-5
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL6/J
Sex: Male
condition: Myocardial infarction
cell type: CD11b+ myeloid cells from heart
time point: mixed sample (non-demultiplexed)
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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 |
Cells were isolated from the heart of mice at 1 (n=5), 3 (n=5) and 5 days post-MI (n=3) as described above. Fc-Block was performed (10µg/ml purified rat anti-mouse CD16/CD32, Biolegend TruStain FcX anti-mouse), after which cells were stained with Fixable Viability Staining e780 (1:1000) and CD11b (M1/70) antibodies: CD11b-BV510 (Day 1 cells, 1:300, BD Biosciences), CD11b-Percp-Cy5.5 (Day 3 cells, 1:300, BD Biosciences) or CD11b-PE (Day 5 cells, 1:300, BD Biosciences) for 25 minutes at 4°C in PBS containing 1% FCS. Cells were washed once, resuspended in 1ml of PBS-1%FCS, pooled and centrifuged once more (8’, 350rcf). Viable/CD45.2 (intravenous)-negative cells labeled with CD11b-BV510, CD11b-Percp-Cy5.5 and CD11b-PE were separately sorted using a FACS Aria III (BD Biosciences). After sorting, cells were washed once in 1ml of PBS-1%FCS (8’, 350rcf). Cells were then separately labeled with hashtag antibodies (Biolegend TotalSeq-A antibodies, Day 1: Hashtag 1, Day 3: Hashtag 2, Day 5: Hashtag 3) and CITE-Seq antibodies (Biolegend TotalSeq-A against mouse Ly6C, Ly6G, CD64, F4/80, CD11c, MSR1, IA/IE, TIM4, CX3CR1, and CCR2). Labeling was performed for 30 minutes at 4°C in 150µl PBS/1%FCS containing 1.5µg of each antibody. Cells were washed with 2ml PBS/1%FCS once (8’, 350rcf), resuspended in PBS supplemented with 0.04% BSA, pooled and centrifuged again (8’, 350rcf). The cells were resuspended in 150µl PBS/0.04% BSA, filtered through a 40µm cell strainer and counted before loading in 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 v3 Chemistry and prepared for CITE-Seq/Hashing according to the manual until the cDNA amplification step in which 1 μl (0.2 μM) ADT PCR additive primer to capture Antibody-derived tags (ADTs) and 1 μl (0.1 μM) HTO PCR additive primer to capture Hashtag oligos (HTOs) were added. After cDNA amplification 60 μl (0.6x) SPRI beads (Beckman Coulter) were added to separate the supernatant fraction that contains the ADT/HTO-derived cDNAs (<180bp) and the bead fraction that contains the mRNA-derived cDNAs (>300bp). The mRNA-derived cDNAs (bead fraction) were processed following the standard 10x Genomics protocol. The ADT/HTO-derived cDNAs (supernatant fraction) were purified twice with 2x SPRI. After the two purifications half amount of the eluted cDNAs were amplified for ADTs and half for hashtags. In the same reactions, the ADTs were indexed using TruSeq Small RNA primers and the hashtags using modified TruSeq DNA primers. The libraries were purified once more with 1.6x SPRI (160 μl). The detailed protocol including the oligo sequences can be accessed here: https://cite-seq.com/protocol
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
CITE-Seq files: ADTs: H10_ADTs_S3_L002_R1_001.fastq.gz
Hashing files: HTOs: H10_HTOs_S4_L002_R1_001.fastq.gz
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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 |
Aug 18, 2020 |
| 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 |
SAMN12441297 |
| SRA |
SRX6647052 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4005128_H10_mm10_barcodes.tsv.gz |
25.4 Kb |
(ftp)(http) |
TSV |
| GSM4005128_H10_mm10_features.tsv.gz |
244.8 Kb |
(ftp)(http) |
TSV |
| GSM4005128_H10_mm10_matrix.mtx.gz |
52.4 Mb |
(ftp)(http) |
MTX |
| GSM4005128_H10_molecule_info.h5 |
726.2 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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