RNA-seq of aortic endothelial cells from endothelial cell-specific Phactr1 knockout mice (apoe null background)

Endothelial cell-enriched aortic RNA was extracted from 8-week-old endothelial cell-specific Phactr1 knockout mice and control mice. All mice were male and apoe null background. Mice were sacrificed by CO2 inhalation and perfused with saline containing 10 U/ml heparin via the left ventricle. Aortic arch and thoracic aorta were isolated and peri-adventitial tissues were removed carefully. A 29-gauge syringe filled with 250 ul of QIAzol lysis reagent (QIAGEN, Hilden, Germany) was inserted into the thoracic aorta end and quickly flushed the aorta (about 1 second). Intima eluates were used for RNA isolation using the miRNeasy mini kit (QIAGEN, Hilden, Germany) following the protocol from the manufacturer. VE-cadherin (endothelial cell marker) and a-smooth muscle actin (a-SMA, smooth muscle cell marker) were used to determine the enrichment of endothelial RNA. Smart-seq2 was performed using EC-enriched aortic RNA. After reverse transcription and cDNA amplification, cDNA was purified using the AMPure XP beads and library was made using the Illumina Nextera kit with indexed S5 and N7 primers according to instructions from the manufacturer. Then the library was size selected between 200-700 bp using the AMPure XP beads, and was examined using qPCR. These libraries were sequenced on the Novaseq 6000 sequencing platform and the reads length were 150 bp with pair-end strategy.