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Sample GSM5738397

Query DataSets for GSM5738397

Status

Public on Dec 18, 2021

Title

pr_RNA_aaUntr_3dpi_ha2

Sample type

SRA

Source name

Untreated animal 2, 3dpi, lung sample for bulk RNA-seq

Organism

Phodopus roborovskii

Characteristics

tissue: Lung
treatment: none
sampling timepoint: 3dpi

Treatment protocol

At 6-10 weeks of age hamsters were infected intranasally as previously described (PMID 32698441, 33271063). Briefly, hamster received 1 x 105 pfu SARS-CoV-2 Variant B1 (BavPat1) intranasally under anesthesia.

Growth protocol

Hamsters were weighted daily and monitored for signs of disease twice-daily. Severely sick animals were euthanized according to defined humane endpoints including body temperature <33 °C, acute respiratory distress or weight loss >20%. Hamsters were selected randomly for all scheduled take-out time points (day 3 and 5 for Roborovski hamsters, day 5 and 7 for Syrian hamsters).
Euthanasia prior analysis occurred by 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.

Extracted molecule

polyA RNA

Extraction protocol

For isolation of single cells lobus caudalis 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.
Bulk and single-cell RNA-sequencing libraries were constructed using commercial kits (New England Biolabs, 10xGenomics), see column "library construction protocol" in the SAMPLES section.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

NEBNext Ultra II Directional RNA Library Prep Kit for Illumina
adapter sequence: GATCGGAAGAGCACACGT

Data processing

For bulk RNA-sequencing, alignments were done using hisat2 (Kim et al, 2015)
Syrian hamster: Sequencing reads were aligned to the MesAur 1.0 version of the Mesocricetus auratus genome using standard parameter, using the Refseq gtf file described in GEO accession number GSE162208
Roborovski hamster: Sequencing reads were aligned to the draft assembly and gtf file described previously (doi: 10.1101/2021.10.02.462569)
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
Seurat R and loom objects were processed as described on the github page https://github.com/Berlin-Hamster-Single-Cell-Consortium/Dwarf-Hamster-Dexamethasone-Antibody
Genome_build: MesAur 1.0
Genome_build: Phodopus roborvskii draft assembly
Genome_build: SARS-CoV-2 MN908947
Supplementary_files_format_and_content: tab-separated values
Supplementary_files_format_and_content: h5 matrix
Supplementary_files_format_and_content: Seurat R object
Supplementary_files_format_and_content: loom object

Submission date

Dec 16, 2021

Last update date

Dec 18, 2021

Contact name

Emanuel Wyler

E-mail(s)

emanuel.wyler@mdc-berlin.de

Phone

+49 30 9406 3009

Organization name

Max Delbrück Center for Molecular Medicine