 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Dec 14, 2021 |
| Title |
scRNA-seq of total mouse aortic cells with CITE-seq and cell Hashing |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
|
| Summary |
We performed single-cell RNA-seq analysis with CITE-seq and cell hashing of total viable aortic cells from Ldlr-/- mice fed a high fat diet for 13 weeks
|
| |
|
| Overall design |
Aortas were collected from Ldlr-/- mice fed a HFD (Altromin, 15% milk fat, 1.25% cholesterol) for 13 weeks and treated with an irrelevant isotype control antibody (100 µg rat IgG2b anti-Phyt1, clone AFRC Mac51, i.p. twice weekly) for 4 weeks before sacrifice. 2 aortas were pooled to generate each sample, and n=5 pools were analyzed. To exclude circulating leukocytes from analysis, mice were injected with 2.5µg anti-CD45.2 APC (clone 104, BioLegend) intravenously 5 minutes before sacrifice to label blood borne immune cells. Aortas were enzymatically digested in RPMI medium 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 with agitation, washed in PBS/1% FCS and passed through a 70µm cell strainer. Cells were labeled for 20 minutes at 37°C with CalceinViolet-AM (BioLegend 425203) according to the manufacturer’s instructions. Cells were washed and resuspended in PBS 1% FCS, plated in round bottom 96 well plates, and incubated for 10 minutes on ice with anti-CD16/32 (Biolegend, Clone 93, 10µg/ml) to block unspecific binding of antibodies to Fc receptor. Cells were subsequently labeled in a final volume of 100µl for 15 minutes at 4°C with anti-CD45.2 Alexa488 (2.5µg/ml, clone 104, BioLegend), anti-Ter119 PE-Cy7 (1µg/ml, clone TER119, BioLegend), 1:1000 Fixable Viability Dye, CITE-seq antibodies (see table), and hashtag antibodies. Each sample was labeled with a specific Hashtag antibody (Hashtag 1 to 5, final concentration 1:200, BioLegend). Cells were washed twice and all the sample were pooled. Viable Ter119negCD45.2-APC(i.v.)negCalceinAM+ cells were sorted using a BD FACS Aria III with a 100µm nozzle. The amount of sorted cells was adjusted so that ~50% of the total cells represent leukocytes (CD45-Alexa488+). Cells were sorted in PBS supplemented with 0.04% BSA, washed and counted in Trypan blue to assess viability (>85%). 18,000 cells were loaded at a concentration of 600 cells/µl in the 10x Genomics Chromium (Single Cell 3’ v3 reagents, 10x Genomics) and scRNA-seq ADT and HTO libraries were prepared.
|
| |
|
| Contributor(s) |
Schäfer S, Saliba A, Zernecke A, Arampatzi P, Cochain C |
| Citation(s) |
34897380 |
| |
| Submission date |
Aug 27, 2021 |
| Last update date |
Dec 16, 2021 |
| Contact name |
Antoine-Emmanuel Saliba |
| E-mail(s) |
emmanuel.saliba@helmholtz-hzi.de
|
| Phone |
+49-931-31-81341
|
| Organization name |
Helmholtz Institute for RNA-based Infection Research
|
| Street address |
Josef-Schneider-Straße 2 / D15
|
| City |
Würzburg |
| ZIP/Postal code |
97080 |
| Country |
Germany |
| |
|
| Platforms (1) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
|
| Samples (1) |
|
| Relations |
| BioProject |
PRJNA758407 |
| SRA |
SRP334511 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE182950_RAW.tar |
383.2 Mb |
(http)(custom) |
TAR (of H5, TAR) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
