title: Illumina HiSeq 2500 sequencing; Bulk RNA-sequencing of FACS isolated human fetal lung, intestine, and kidney endothelial cells
library_construction_protocol: De-identified human fetal lung, intestine, and kidney tissue was obtained from the University of Washington Laboratory of Developmental Biology. Tissue was shipped overnight in Belzer-UW Cold Storage Solution (ThermoFisher, NC0952695) with cold packs (Menon et al., 2018; Miller et al., 2020). Primary human tissue was dissociated into single cell suspensions for FACS isolation of ECs according to a previously published protocol (van Beijnum et al., 2008). Single cell suspensions were passed through a 70µm filter, pelleted, and resuspended in staining buffer comprised of PBS with 1x PenStrep, 2 µM EDTA, and 2% FBS. Cells were stained with CD144-APC and CD31-PE or corresponding isotype controls for 30 minutes on ice in a total volume of 100 µl per 1x107 cells. Cells were washed three times in excess staining buffer accompanied by centrifugation at 300xg for 5 mins between washes. Cells were resuspended in staining buffer with DAPI (0.2 µg/ml). Antibody stained samples and controls were analyzed and sorted on a FACSAria III cell sorter (BD), and analysis was performed using BD FACSDiva software. Any post-acquisition analysis was performed using FlowJo. Cells were sorted into staining buffer, and after sorting cells were snap frozen and stored at -80 oC prior to RNA isolation. RNA was isolated from snap frozen cell pellets using the RNeasy Mirco Kit (74004, Qiagen), according to manufacturer's guidelines. cDNA libraries were prepared using the SMARTer Stranded Total RNA-Seq Kit v2- Pico Input (634412, Takara) by the University of Michigan Advanced Genomics Core according to manufacturer's guidelines.