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| Status |
Public on May 16, 2022 |
| Title |
Experiment 2, GEX_Pool2_part1 |
| Sample type |
SRA |
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| Source name |
multiplexed sample
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| Organism |
Mesocricetus auratus |
| Characteristics |
first vaccination: Multiplexed sample (mixed treatments)
second vaccination: Multiplexed sample (mixed treatments)
sampling timepoint: 2dpi
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| Treatment protocol |
21 days following primary vaccination or 14 days following booster vaccination, hamsters were infected intranasally as previously described (PMID 32698441, 33271063). Briefly, hamster received 1 x 105 pfu SARS-CoV-2 Variant Delta. [SARS-CoV-2, Human, 2021, Germany ex India, 20A/452R (B.1.617)] intranasally under anesthesia.
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| Growth protocol |
Hamsters were vaccinated intramuscularly with either an experimental adenovirus vector vaccine containing the whole SARS-CoV-2 spike (5 _ 108 infectious units (IFU) per animal) (PMID: 34835096), or BNT162b2 (5 ug per animal) or intranasally with 1x105 pfu of the live-attenuated SARS-CoV-2 vaccine candidate sCPD9 (PMID: 34320400, PMID: 34851677). For the prime-boost experiment, immunity was boosted by a second vaccination with the respective vaccines at the same dose 21 days after primary vaccination. In, one group, animals were primed with BNT162b2 and boosted with sCPD9, all other groups received a homologues boost.
Hamsters were weighted daily and monitored for signs of disease twice-daily. Hamsters were selected randomly for all scheduled take-out time points (day 2 and 5).
Euthanasia prior analysis occurred by cervical dislocation exsanguination under anesthesia as previously described (PMID 32698441, 33271063). Peripheral blood was collected in EDTA-coated syringes and lung lobes were collected for follow-up analyses, in which the left lobe was used for histopathology, the right caudal lobe for single-cell analysis, the right cranial lobe for virological assessments and the right medial as backup to repeat virological analyses as needed.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Bulk and single-cell RNA-sequencing libraries were constructed using commercial kits:
10xGenomics Chromium v3.1 single cell 3' gene expression kit
For isolation of single cells, the caudal lobe of the right lung was removed and placed in storage medium (1× PBS, 0.5 % BSA) until further processing. Storage and isolation media contained 2 µg/mL ActinomycinD. Tissues and cells were always spun at 350 x g for 6 minutes at 4 °C. Mechanical dissociation of lung lobes was achieved by clapping motions with tweezers for 2 minutes in enzymatic 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 followed by 30 minutes incubation at 37°C and 5 % CO2.
Further tissue dissociation was achieved by repeated pipetting of digested lung tissue with 5 mL pipettes and passing of cell suspension with plungers through 70 µm cell strainers. Cells were pelleted from suspension by centrifugation and resuspended in red blood cell lysis buffer (BioLegend). Lysis reaction was stopped and cells washed with excessive volumes of PBS/BSA. Cells were centrifuged, resuspended in low-BSA buffer (1× PBS, 0.04 % BSA) and counted via hemocytometer and trypan blue.
Then, 1,000,000 lung cells per sample were subjected to Cell Multiplexing Oligo (CMO) labeling according to the manufacturers’ instructions (3ʹ-CellPlex-Kit-Set-A, 10x Genomics). Labelled cells from 12 samples were pooled, filtered (40 µm) and counted. Pooled cells were adjusted to a final concentration of ~1,600 cells / μL and filtered with 40 µm FloMi filters (Merck) suitable for low volumes prior subjection to scRNA sequencing.
For Lung RNA Bulk sequencing the lobus medialis of the right lung lobe was removed carefully and homogenzied in Trizol reagent using a bead mill procedure (Speedmill, Analytic Jena), RNA was extracted according to the manufacturer’s instructions.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
exp2_GEX_Pool2_part1
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| Data processing |
The Mesocricetus auratus genome MesAur 2.0 was downloaded from https://www.ncbi.nlm.nih.gov/genome/11998. The GFF file was converted to GTF, the generic gene names (LOCxxx) replaced with mouse-derived official gene symbols for several key genes, and 3'-UTRs extended as described previously (doi: 10.1101/2021.10.02.462569). Then, the SARS-CoV-2 genome annotation was merged.
For bulk RNA-sequencing, alignments were done using hisat2 (Kim et al, 2015)
For single-cell RNA-sequencing data, fastq files were processed using a the mergend MesAur2.0/SARS-CoV-2 genome with the GTF file provided here by applying CellRanger 6.0.2 with standard parameters
For single-cell RNA-sequencing data, fastq files were processed using a merged Roborvski hamster/SARS-CoV-2 genome by applying CellRanger 6.0.2 with standard parameters
Assembly: MesAur 2.0
Assembly: SARS-CoV-2 MN908947
Supplementary files format and content: GTF file
Supplementary files format and content: tab-separated values
Supplementary files format and content: h5 matrix
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| Submission date |
Apr 11, 2022 |
| Last update date |
May 17, 2022 |
| Contact name |
Emanuel Wyler |
| E-mail(s) |
emanuel.wyler@mdc-berlin.de
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| Phone |
+49 30 9406 3009
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| Organization name |
Max Delbrück Center for Molecular Medicine
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| Department |
Berlin Institute for Medical Systems Biology
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| Lab |
RNA Biology and Posttranscriptional Regulation
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| Street address |
Robert Roessle Str 10
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| City |
Berlin |
| ZIP/Postal code |
13125 |
| Country |
Germany |
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| Platform ID |
GPL28997 |
| Series (1) |
| GSE200596 |
A live attenuated vaccine confers superior mucosal and systemic immunity to SARS-CoV-2 variants |
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| Relations |
| BioSample |
SAMN27518118 |
| SRA |
SRX14814201 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6038429_exp2_Pool2_part1_part1_LU_A1_sample_feature_bc_matrix.h5 |
2.9 Mb |
(ftp)(http) |
H5 |
| GSM6038429_exp2_Pool2_part1_part1_LU_A2_sample_feature_bc_matrix.h5 |
3.2 Mb |
(ftp)(http) |
H5 |
| GSM6038429_exp2_Pool2_part1_part1_LU_A3_sample_feature_bc_matrix.h5 |
4.1 Mb |
(ftp)(http) |
H5 |
| GSM6038429_exp2_Pool2_part1_part1_LU_B1_sample_feature_bc_matrix.h5 |
2.8 Mb |
(ftp)(http) |
H5 |
| GSM6038429_exp2_Pool2_part1_part1_LU_B2_sample_feature_bc_matrix.h5 |
3.5 Mb |
(ftp)(http) |
H5 |
| GSM6038429_exp2_Pool2_part1_part1_LU_B3_sample_feature_bc_matrix.h5 |
3.3 Mb |
(ftp)(http) |
H5 |
| GSM6038429_exp2_Pool2_part1_part1_LU_X1_sample_feature_bc_matrix.h5 |
3.3 Mb |
(ftp)(http) |
H5 |
| GSM6038429_exp2_Pool2_part1_part1_LU_X2_sample_feature_bc_matrix.h5 |
4.1 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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