Pulmonary_EC_01_RNAseq
For isolation of lung endothelial cells, lungs were removed, cut into small pieces and incubated in enzyme solution at 37  C for 30 minutes. A single cell suspension was obtained by repetitive pipetting and filtered through a sterile 100  m mesh size cell strainer prior to FACS. Cell suspensions were labelled by the far-red fluorescent dye Draq5 at a dilution of 1:2000. Using FACS (S3 Cell Sorter, BioRad) endothelial cells were identified by Draq5 positivity, low forward scatter area and GFP positivity and sorted into RLT Plus buffer (Qiagen) containing 10  l/ml  -mercaptoethanol for isolation of RNA. Sorting was carried out at 4  C. Total RNA was isolated from from purified endothelial cells using an AllPrep DNA/RNA Micro Kit (Qiagen) according to the manufacturer s instructions. DNA was isolated to ensure DNA-free RNA yields. The quality was assessed in a Bioanalyzer (2100 Bioanalyzer, Agilent). For preparation of amplified cDNA the Ovation RNA-Seq System V2 Kit (NuGEN) was used according to the provided user guide. The cDNA was fragmented to approximately 350 bp fragments using a sonication device (Bioruptor, Diagenode) and 100 ng were used for library construction with the NEB Next Ultra DNA Library Prep Kit for Illumina (New England BioLabs) and amplified by fluorescence-controlled PCR. Size selection was performed with AMPure XP Beads (Beckmann Coulter) according to the manual of the kit. Quality and mean fragment size of library samples were assessed in a Bioanalyzer.
SUB3407571
Pulmonary_EC_01