Mus musculus
Protocols: Mice were sacrificed at age 6-10 weeks and tissue from lung or excised tumors on day 23 were disassembled and digested for further stromal cell or immune cell isolation and scRNA sequencing analysis. To validate the differentiation process of fibroblasts in murine lung cancer, fatemapping experiments were performed using the inducible tetracycline transactivase (Tta)-based Ccl19-Tta mouse model in combination with single-color R26R-EYFP (Ccl19-iEYFP) mice. Ccl19-iEYFP mice were treated with doxycycline between day 15 and 23, enabling progeny of day 15 progenitors to be analysed on day 23. Then, single cell RNA-sequencing of fate mapped Ccl19-iEYFP+ cells was performed. For enzymatic digestion of disaggregated lung tissue from tumors or excised tumors (day 23), 1 mg/ml Dispase (Roche), 1 mg/ml Collagenase Type II (Gibco) and 25 μg/ml DNaseI (AppliChem) in combination with gentleMACS-based mechanical disruption (Miltenyi Biotech) were used. After 30 minutes incubation at 37°C, cell suspensions were washed with PBS containing 0.5% FCS and 10 mmol/L EDTA. Enrichment of stromal cells was achieved by depleting hematopoietic and erythroid cells using MACS anti-CD45 and anti-Ter119 microbeads (Miltenyi Biotec). The flow-through was centrifuged and single cell suspension was stained for further flow cytometric analysis or cell sorting. The unbound single cell suspension was stained for cell sorting with a BD FACS Melody 2-way or 4-way cell sorter (BD Biosciences) and run on the 10x Chromium analyzer (10X Genomics). For the isolation of immune cells, tissues were gently smashed through a mesh using a syringe plunger and washed with PBS containing 0.5% FCS and 10 mmol/L EDTA before staining for flow cytometric analysis or cell sorting. cDNA library generation was performed following the established commercial protocol for Chromium Next GEM Single Cell 3ʹ GEM, Library & Gel Bead Kit v3/3.1 Chemistry.
ENA first public
2024-10-01
External Id
SAMEA115120934
INSDC center name
Institute of Immunobiology, Kantonsspital St. Gallen, St.Gallen, Switzerland
INSDC last update
2024-01-20T10:54:12Z
INSDC status
public
Submitter Id
E-MTAB-13708:Sample 6
age
6 to 10
broker name
ArrayExpress
cell_type
fibroblast
collection date
not collected
common name
house mouse
developmental stage
adult
genotype
Tg(Ccl19-tTA)+ x R26-EYFP++
geographic location (country and/or sea)
not collected
individual
mixed pool of 4 to 10 individuals
isolate
not applicable
mcov treatment
No
organism part
lung unaffected
sample name
E-MTAB-13708:Sample 6
scientific_name
Mus musculus
sex
mixed
strain
C57Bl/6N