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| Status |
Public on Jun 10, 2024 |
| Title |
High-dose infected day 3 Hamster 1 |
| Sample type |
SRA |
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| Source name |
lung
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| Organism |
Phodopus roborovskii |
| Characteristics |
tissue: lung
infection: SARS-CoV-2 Variant B.1
virus dose: 1x10^5 pfu
time: 3 dpi
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| Treatment protocol |
10- to 12-week-old male and female Syrian and Roborovski hamsters were infected intranasally with 1x10^4 or 1x10^5 plaque-forming units (pfu) SARS-CoV-2 (variant B1, isolate BetaCoV/Germany/BavPat1/2020) under anesthesia as previously described (PMID:34320400). To prevent any prolonged suffering, the hamsters were clinically examined twice daily. Animals with body weight loss >15% for more than 48 hours were euthanized according to the animal use protocol. For Roborovski hamsters, naive animals (n = 3) and animals at 2, and 3 days post infection (n = 3 each) were randomly selected. Euthanasia was performed by cervical dislocation and exsanguination under anesthesia as previously described (PMID:32698441). Among other materials, all lung lobes were collected for subsequent analyses. Specifically, the left lobe was used for histopathology, the right caudal lobe for single-cell analysis, the right cranial lobe for virological measurements, and the right middle lobe for bulk RNA and proteomic analysis as described (PMID:34381043).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Established cell isolation protocols were modified to comply with BSL3 facility regulations. For single cell isolation, the caudal lobes of the right lung were stored in 1× PBS, 0.5% BSA containing 2 µg/ml actinomycin D. The lobes were dissociated mechanically and enzymatically. Tissue was first disrupted with forceps for 2 minutes in specific digestion medium (3.4 mg/mL collagenase Cls II (Merck), 1 mg/mL DNase I (PanReac AppliChem) in 2 mL Dispase medium per lung lobe (Corning), 50 caseinolytic units/mL) and then enzymatically digested at 37°C and 5% CO2 for 30 minutes. The cell suspension was further dissociated by pipetting, and filtered through 70 µm cell strainers. The suspensions were centrifuged at 350 g for 6 minutes at 4 °C, and the pellets were subjected to erythrocyte lysis by resuspension in appropriate buffer (BioLegend). The reaction was stopped by washing with PBS/BSA buffer and the cells were centrifuged. Cells were resuspended in low BSA buffer (1 × PBS, 0.04% BSA) and then filtered through 40 µm FloMi filters (Merck). Cell number and viability were determined microscopically using trypan blue.
Sequencing libraries were generated using the 3’ Chromium Next GEM Single Cell 3’ Reagent kit (10x Genomics) according to the manufacturer’s instructions, and sequenced on a NovaSeq 6000 device (Illumina).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Alignment, demultiplexing, UMI quantification, and cell-barcode selection (basic filtering) was done with cellranger v6.1.1 (10X Genomics).
Cells with less than 250 genes expressed were filtered out. After cell type identification, we filtered each cell type using the respective median UMI count as lower threshold prior to final cell type annotation.
Annotation and filtering of doublets using scrublet (v9.2.3).
Count normalization using scanpy.pp.normlize_total from scanpy (v1.9.1).
Log1p transform using scanpy.pp.log1p from scanpy (v1.9.1).
Assembly: doi:10.1093/gbe/evac100 (PMID: 35778793)
Supplementary files format and content: AnnData formatted sparse count matrix as AnnData (v0.8.0) .h5 files with all samples merged and processed.
Supplementary files format and content: Cellranger formatted sparse count matrices as .h5 file per sample.
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| Submission date |
Aug 17, 2023 |
| Last update date |
Jun 10, 2024 |
| Contact name |
Stefan Peidli |
| Organization name |
European Molecular Biology Laboratory
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| Department |
Genome Biology
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| Lab |
Huber
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| Street address |
Meyerhofstraße 1
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| City |
Heidelberg |
| State/province |
BW |
| ZIP/Postal code |
69117 |
| Country |
Germany |
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| Platform ID |
GPL31105 |
| Series (1) |
| GSE241133 |
Single-cell-resolved interspecies comparison shows a shared inflammatory axis and a dominant neutrophil-endothelial program in severe COVID-19 |
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| Relations |
| BioSample |
SAMN37041987 |
| SRA |
SRX21405176 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7717100_cellranger_pr-hd-D3-Z1_filtered_feature_bc_matrix.h5 |
11.3 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
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