organism: Mus musculus
description: Protocols: Lungs were divided into three portions, each incubated in serum-free medium with dispase (1:300), collagenase I (2 mg/mL), and DNase I (1:1000). Tissue was finely minced, brought to 1 mL with enzyme mix, and digested at 37°C with shaking (800 rpm) in three 10-minute cycles, followed by a final 20-minute digestion. Digests were pooled into cold medium with 10% FBS to stop enzyme activity, filtered through a 70 µm mesh, and centrifuged (500 × g, 10 min, 4°C). Pellets were treated with ACK lysis buffer to remove erythrocytes, incubated on ice for 5 minutes, washed with medium containing 10% FBS, and centrifuged again (500 × g, 5 min, 4°C). The cell pellet was resuspended in 10% FBS medium containing fluorescently conjugated antibodies: anti-mouse CD45-Pacific Blue (PB; Biolegend), CD31-PE (eBioscience), and EpCAM-APC (CD236; eBioscience), each at a final dilution of 1:400. Unstained and single-color controls were included. Cells were incubated on ice for 15–30 minutes. Following staining, cells were washed with 10 mL of 10% FBS medium and centrifuged again (500 × g, 5 minutes, 4°C). The pellet was resuspended in 200–500 µL of HBSS with 3% FBS. The CD31⁺ population was FACS sorted directly into RLT lysis buffer; EpCAM⁺ cells served as epithelial controls. Mice were euthanized, and the thoracic cavity was opened to expose the heart and lungs. Lungs were perfused via the heart with 3–5 mL of HBSS containing dispase (1:300), causing visible blanching, and the inferior vena cava was transected to aid drainage. Lungs and heart were excised, rinsed in HBSS, and the heart was removed. Total RNA was isolated using micro kit (Qiagen) from 50 000 FACS-sorted cells. Libraries were prepared using Smarter stranded total RNA-Seq kit v2 - pico Input mammalian PN-634413 (Takara).
attributes.tag: ENA first public
attributes.value: 2025-08-22
attributes.tag: INSDC center name
attributes.value: Institute of Molecular Genetics of the Czech Academy of Sciences
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-15355:s_839
attributes.tag: age
attributes.value: 12
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: collection date
attributes.value: not collected
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: genotype
attributes.value: RH/RH
attributes.tag: geographic location (country and/or sea)
attributes.value: not collected
attributes.tag: individual
attributes.value: H
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: pulmonary endothelial cells
attributes.tag: sample name
attributes.value: E-MTAB-15355:s_839
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: female
attributes.tag: strain
attributes.value: C57BL6/N