title: Transcriptomic profile of valvular endothelial cells in normal and high glucose conditions, interacted with human monocytes (THP-1)
design: Transcriptomic profile of valvular endothelial cells in normal and high glucose conditions, interacted with human monocytes (THP-1)
library_construction_protocol: VECs after interaction with monocytes in NG or HG conditions, monocytes were removed by washing with PBS and total RNA from VECs was isolated with TRIzol reagent and Phasemaker tubes. VECs in NG or HG media were used as controls. For each condition, 3 biological replicates were collected in 3 independent experiments.  Primary human valvular endothelial cells (VEC) were sourced from aortic valve leaflets extracted from patients undergoing aortic valve replacement surgery. VEC population was subsequently cultured in EGM-2 media supplemented with 10% FBS and 1% penicillin/streptomycin in normal glucose concentration (NG – 5 mM glucose) or in high glucose concentrations (HG – 33 mM glucose).  Monocyte‐like cell line THP‐1 was grown in suspension in the RPMI 1640 culture medium containing 10% FCS and were split 1:5, twice a week.  To set up the VEC-monocyte co-culture, we first incubated the monocytes for 72 hours in either normal glucose (NG) media (Mo-NG) or high glucose (HG) media (Mo-HG). Then, we introduced 1.5x10^6 monocytes/ml to the VEC monolayer for 2 hours. For this addition, Mo-NG was added to VECs in NG media, and Mo-HG was added to VECs in HG media, matching each monocyte type with its respective media. As controls, we used VECs in NG and VECs in HG media without monocytes. Total RNA was extracted using the manufacturer's recommended protocol: The Phasemaker tubes were centrifuged for 30 seconds at 12,000-16,000 × g before use. Samples were lysed and homogenized in TRIzol Reagent. After adding chloroform, the mixture was centrifuged to separate into three layers: a clear upper aqueous layer containing RNA, an interphase, and a red lower organic layer with DNA and proteins. The Phasemaker tubes facilitated the transfer of the aqueous layer by creating a barrier between the upper and lower phases. RNA was then precipitated from this aqueous layer using isopropanol. Following precipitation, the RNA was washed to remove any impurities and subsequently resuspended for downstream applications. RNA concentration and purity were quantified using the NanoDrop 1000 (ThermoFisher, USA).  Library construction, quality control and sequencing were performed by Novogene (Cambridge, UK). A total amount of 1 µg RNA per sample was used as input for RNA analysis.  Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using dUTP. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection.