characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2022-05-20T16:17:49Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2022-08-23T18:21:01Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA110039406
characteristics.characteristic: External Id
characteristics.text: University of Liege, GIGA Institute, Laboratory of Cellular and Molecular Immunology
characteristics.characteristic: INSDC center name
characteristics.text: 2022-05-20T16:17:49Z
characteristics.characteristic: INSDC first public
characteristics.text: 2022-08-23T18:21:01Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS12023650
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-6902:Sample 3
characteristics.characteristic: Submitter Id
characteristics.text: 10
characteristics.unit: week
characteristics.characteristic: age
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: neutrophil
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CL_0000775
characteristics.characteristic: cell type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: To obtain single-lung-cell suspensions, lungs were perfused with 10 ml of PBS through the right ventricle, cut into small pieces, and digested for 1 h at 37°C in HBSS containing 1 mg/ml collagenase A (Roche) and 0.05 mg/ml DNase I (Roche). For droplet-based single cell RNA sequencing (scRNA-seq), single-lung-cell suspensions were obtained from perfused lungs of PBS-injected, LPSlow (100 ng)-treated and LPS high (10 µg)-treated mice (n=3/group). Neutrophils were first enriched by negative selection using a magnetic-activated cell sorting (MACS, Neutrophil Isolation kit, Miltenyi Biotec) and then sorted as Cd45.2+ cells using nozzle 100 at 3000 events/second for 5 minutes (FACS Aria III). Cell preparations were centrifuged at 300g for 5 min and resuspended in calcium- and magnesium-free PBS containing 400µg/ml bovine serum albumin (BSA, Sigma-Aldrich). Trypan blue-treated cells were examined under the microscope for counting and viability testing. Viability was above 95% for all three samples. Isoflurane-anesthetized BALB/c wild-type mice were exposed intranasally to low (100 ng) or high (10 µg) doses of LPS (LPS from Escherichia coli O55:B5, Sigma-aldrich). Vehicle-administered mice were instilled i.n. with 50 µl sterile PBS. One day later (Day 0), mice were euthanized, lungs were resected and lung single-cell suspensions were obtained. Cellular suspensions were loaded on the Chromium™ Controller (10x Genomics, Pleasanton, CA, USA) in order to generate Gel Bead-In-EMulsions (GEMs). Sequencing libraries were generated by using the Chromium™ Single Cell 3’ Reagent Kits v2 (10x Genomics, Pleasanton, CA, USA) following manufacturer’s instructions. GEM-RT was performed in a Veriti© 96-Well Thermal Cycler (Fisher Scientific, Merelbeke, Belgium). After RT, GEMs were broken and the cDNAs were cleaned up with DynaBeads MyOne Silane Beads (Fisher Scientific, Merelbeke, Belgium). cDNAs were then amplified in a Veriti© 96-Well Thermal Cycler. According to the expected cell recovery (based on a 60% recovery of total loaded cells), number of amplification cycles was set to 14. Amplified cDNA products were cleaned up using SPRIselect Reagent kit (Beckman Coulter, CA, USA), after what purified cDNAs were quality controlled and quantified using an Agilent High Sensitivity DNA kit on a 2100 Bioanalyser (Agilent, CA, USA). Illumina’s P5, P7 and Read2 primers, as well as Sample Index were then added to generate sequencing libraries following Chromium™ Single Cell 3’ Reagent Kits v2 protocol. Steps were as follows: (1) enzymatic fragmentation, end repair and A-tailing, (2) Double Sided Size Selection using SPRIselect reagent, (3) adaptor ligation, (4) post ligation cleanup with SPRIselect reagent, (5) Sample index PCR (number of cycles set to 14) and (6) Double Sided Size Selection using SPRIselect reagent. The barcoded sequencing libraries were quality controlled using an Agilent High Sensitivity DNA kit on a 2100 Bioanalyser and quantified by quantitative PCR (KAPA Biosystems Library Quantification Kit for Illumina platforms).
characteristics.characteristic: description
characteristics.text: wild type genotype
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0005168
characteristics.characteristic: genotype
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_10090
characteristics.characteristic: organism
characteristics.text: lung
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/UBERON_0002048
characteristics.characteristic: organism part
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: female
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000383
characteristics.characteristic: sex
characteristics.text: BALB/c
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0000602
characteristics.characteristic: strain
characteristics.text: Sample 3
characteristics.characteristic: title