RNA-seq of aortic endothelial cells from Phactr1 knockout mice

Endothelial cell-enriched aortic RNA was extracted from global Phactr1 knockout male mice and control wildtype mice of 10-12 weeks old. 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. RNA-seq analysis was conducted using EC-enriched aortic RNA. Briefly, the libraries were constructed using TruSeq Stranded mRNA LTSample Prep Kit (Illumina, San Diego, CA, USA) according to instructions from the manufacturer. Then these libraries were sequenced on the Illumina HiSeq X Ten sequencing platform and 125bp/150bp paired-end reads were generated.