title: Expression profiling of endothelial cells from old (12 months) and very old (18 months) homozygous Jak2 R1063H mice and wild-type controls
library_construction_protocol: 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).