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| Status |
Public on Jul 21, 2022 |
| Title |
scRNA-seq of mouse cardiac CD45+ cells with CITE-seq and cell Hashing II |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
We performed scRNA-seq and CITE-seq of CD45+ cells extracted from the steady state mouse heart, and at 5 days after myocardial infarction in wildtype and Trem2 deficient mice
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| Overall design |
Myocardial infarction (MI) was induced by permanent coronary artery ligation in Trem2+/+ (wildtype) and Trem2-/- mice. Non-infarcted wildtype mice (n=2), non-infarcted Trem2-/- mice (n=3), wildtype mice at day 5 after myocardial infarction (n=5) and Trem2-/- mice at day 5 after myocardial infarction (n=5received an i.v. injection of 2.5µg anti-CD45.2 APC (clone 104, Biolegend, cat. # 109814) under isoflurane anesthesia. The heart was perfused via intracardiac injection of PBS, excised, the right ventricle removed, and the infarct, peri-infarct area, and adjacent viable myocardium were collected, weighed and digested for 1 hour at 37°C under agitation in RPMI containing 450U/ml collagenase I (Sigma-Aldrich, #C0130), 125U/ml Collagenase XI (Sigma-Aldrich C7657), 60U/ml Hyaluronidase (Sigma-Aldrich, H3506) , 60U/ml DNAse (Roche #11284932001). Cells were washed twice in MACS buffer (PBS+0.5% BSA+2mM EDTA) and incubated for 5 minutes on ice with TruStain FcX™ (anti-mouse CD16/32, Biolegend #101320, 10µg/ml). Afterwards, cells were incubated with anti-VSIG4-PE (clone NLA-14, final concentration 2µg/ml, ThermoFisher #12-5752-82), anti-LYVE1-Biotin (clone ALY7, 5µg/ml, ThermoFisher #13-0443-82), mouse CD45 microbeads (Miltenyi, 130-052-301, 1:10 final concentration), anti-mouse CD45.2-AlexaFluor488 (Trem2+/+ heart samples, clone 104, 2µg/ml, Biolegend) or anti-mouse CD45.2-Alexa700 (Trem2-/- heart samples, clone 104, 2µg/ml, Biolegend). After 10’ incubation at 4°C, TotalSeq-A hashtag antibodies were added as follows: Hashtag 1-2: WT no MI; Hashtag 3, 4 and 15: Trem2-/- no MI; Hahstag 5, 6, 7, 8, 9: wildtype MI day 5; Hashtag 10, 11, 12, 13, 14: Trem2-/- MI day 5. Cells were incubated for an additional 15 minutes at 4°C, washed twice in MACS buffer, pooled, and magnetic selection was performed using LS Columns (Miltenyi (# 130-042-401) and MidiMACS separators (Miltenyi # 130-042-302). Sorted CD45+ cells were washed twice with PBS containing 0.04% BSA (Sigma-Aldrich A1595), and incubated for 25’ at 4°C in PBS/0.04% FCS containing Fixable Viability Dye eFluor™ 780 (ThermoFisher #65-0865-14, 1:1000) and anti-mouse TotalSeqA-Antibodies. Cells were washed with PBS/0.04% FCS, resuspended and sorted using a BD FACS Aria III with a 100µm nozzle. Cells from steady state hearts (~50% Trem2+/+ and ~50% Trem2-/-) and from day 5 post-MI hearts (~50% Trem2+/+ and ~50% Trem2-/-) were sorted separately and pooled post-sort so that steady state and day 5 post-MI cells represent ~30% and ~70% of the total cell pool, respectively. 20,000-25,000 cells were loaded in the 10x Genomics Chromium in duplicate lanes.
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| Contributor(s) |
Saliba A, Arampatzi P, Cochain C |
| Citation(s) |
35950218 |
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| Submission date |
Mar 03, 2022 |
| Last update date |
Oct 21, 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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| Platforms (1) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
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| Samples (6)
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| Relations |
| BioProject |
PRJNA812514 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE197853_G8_2_feature.csv.gz |
976 b |
(ftp)(http) |
CSV |
| GSE197853_G8_2_filtered_feature_bc_matrix.tar.gz |
117.2 Mb |
(ftp)(http) |
TAR |
| GSE197853_G9_2_feature.csv.gz |
976 b |
(ftp)(http) |
CSV |
| GSE197853_G9_2_filtered_feature_bc_matrix.tar.gz |
93.3 Mb |
(ftp)(http) |
TAR |
| GSE197853_README_G8_2_G9_2.txt |
267 b |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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