title: GSM8123775: LEC Tie1 WT Replicate2; Mus musculus; RNA-Seq
library_construction_protocol: Skin samples were collected from E18.5 embryo (n=2 for each group) and cut into small pieces . Each skin sample was digested in the enzyme cocktail that contains dispase II, collagenase type2 and DNase I at 37 °C for 1 hour with rotation. In the middle of the digestion, skin pieces were passed through a 1 mm pipet tip 3-5 times to further dissociate the skin. After incubation, a syringe plunger and 100 μm cell strainer were used rub and filter the digested tissue into a 50 ml flacon tube. The strainer was thoroughly rinsed with cold PBS to recover the digested cells. The 50 ml tubes were spun at 300 g x 10 min. Cell pellets were collected by centrifuge at 300 g for 10 min,  and red blood cells were lyzed using RBC Lysis Buffer (Cell Signaling, #46232). Cells were washed with PBS, stained with fixable viability dye and labeled with fluorochrome-labeled antibodies against CD45, CD31 and LYVE1 in FACS buffer. Live single cells that are CD45 negative and CD31 positive were defined as endothelial cells. Sorting was performed on a FACS Aria III flow cytometer with the FACSDiva software (BD Biosciences) using a 100 µm nozzle. LYVE1-high endothelial cells were sorted directly into lysis buffer for RNA extraction using the RNeasy Plus Micro kit (Qiagen, 74034). The quality of isolated RNA was checked using the Agilent 2100 Bioanalyzer. Library prep is based on SMART-Seq v4 chemistry, which uses oligo(dT) to prime for full-length cDNA generation.