title: Illumina HiSeq 2500 paired end sequencing; Bulk RNA-sequencing of VE-Cadherin+/CD45- mouse aorta endothelial cells
design: Bulk RNA-sequencing of VE-Cadherin+/CD45- mouse aorta endothelial cells
library_construction_protocol: Mouse aorta was first digested for 1 hour at 37°C in 1 mg/mL collagenase I (Gibco, Life Technologies), 1 mg/mL dispase (Gibco, Life Technologies), 150 µg/mL DNase-I (Sigma-Aldrich) before passing the suspension through a 70 µmol/L cell strainer. Lineage+ cells were then depleted from tumor cell suspensions via MACS cell separation according to the manufacturer's instructions (Miltenyi Biotech). Cell number and viability for each sample was assessed by using 0.4% Trypan blue solution and a hemocytometer. Single-cell suspensions were then used for flow sorting or analysis by flow cytometry. ENSG ids were converted to gene symbols using tximport. RNA was extracted from sorted endovascular progenitor (EVP), transit amplifying (TA), and definitive differentiated (D) cells using a QIAGEN mini kit (Qiagen) according to the manufacturer's instructions. RNA quality and concentration was assessed by using the Agilent 2100 Bioanalyser (Agilent Technologies). Five to 100 ng of RNA was used for cDNA synthesis using the Superscript III Reverse Transcription Kit (Invitrogen).  Total isolated RNA of appropriate RNA integrity number (>4) was prepared using the TruSeq stranded total RNA library preparation kit as per the manufacturer's instructions.
attributes.tag: Experimental Factor: phenotype
attributes.value: CD31_hi/VEGFR2_hi