Renal_EC_03_RNAseq
For isolation of kidney endothelial cells one kidney was excised after cervical dislocation. After removal of the capsule, kidneys were cut by scissors and incubated with enzyme solution at 37  C for 30 minutes1. 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
Renal_EC_03