title: Sample 3
organism: Mus musculus
description: Protocols: Mice were sacrificed at age 8-10 weeks and lungs were harvested for further stromal cell or immune cell isolation and scRNA sequencing analysis. For cell preparation tissues were cut into small pieces. M-CoV immunization protocol: Mice were infected intranasally with 5 × 104 plaque-forming units (PFU) of M-CoV-A59 of M-CoV-GFP . Lungs were collected at 36 hours after infection for further processing for single cell RNA-sequencing Cell isolation from mouse lungs was performed according to standardized protocols. Mice were euthanized at the indicated time points and immediately perfused with PBS. Lungs were transferred into a 6-well plate containing RPMI 1640 medium (Sigma-Aldrich) supplemented with 4 U/mL elastase (Worthington), 1 U/mL Dispase II (Roche) and 10 μl/mL DNAse I (Applichem) and incubated for 45 minutes at 37°C. Lung tissue was then mechanically disrupted and washed in cold RPMI. Tissues were subject to a second enzymatic digestion in RPMI 1640 medium supplemented with 25 μg/mL Liberase TM (Roche) and 0.25 μg/mL DNAse I and incubated for 30 minutes at 37 °C. Any larger tissue fragments were dissociated mechanically followed by washing of the cell strainer with MACS buffer (PBS, 2% FCS, 2mM EDTA (Sigma-Aldrich)). Stromal and hematopoietic cell fractions were enriched using MACS anti-CD45 microbeads (Miltenyi Biotec). Cell sorting was performed on a BD FACSMelody™ cell sorter using BDChorus software. For discrimination between live and dead cells, Seven-aminoactinomycin D (Sigma-Aldrich) was added prior to sorting. Sorted cells were passed through the 10x Chromium (10x Genomics) system and cDNA libraries were generated according to the manufacturer's recommendations (Chromium Single Cell 3′ Reagent Kit (version 3 chemistry)). cDNA library generation was performed following the established commercial protocol for Chromium Single Cell 3' Reagent Kit (v3 Chemistry).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2023-06-01
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-06-01
attributes.tag: External Id
attributes.value: SAMEA110136675
attributes.tag: INSDC center alias
attributes.value: Institute of Immunobiology, Kantonsspital St. Gallen, St. Gallen, Switzerland
attributes.tag: INSDC center name
attributes.value: Institute of Immunobiology, Kantonsspital St. Gallen, St. Gallen, Switzerland
attributes.tag: INSDC first public
attributes.value: 2023-06-01T00:15:14Z
attributes.tag: INSDC last update
attributes.value: 2023-06-01T00:15:14Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-11781:Sample 3
attributes.tag: age
attributes.value: 8 to 10
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_type
attributes.value: non-hematopoietic cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: immunophenotype
attributes.value: CD45-; GFP-
attributes.tag: individual
attributes.value: pool of 4 to 8 individuals
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: lung
attributes.tag: sample name
attributes.value: E-MTAB-11781:Sample 3
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: female
attributes.tag: strain
attributes.value: C57BL/6N