Illumina NovaSeq 6000 paired end sequencing; Aortic valve transcriptome in CHIP, non-CHIP and non-calcific controls.
Aortic valve leaflets removed during surgery were immediately immersed in RNAlater (ThermoFisher Scientific, Waltham, Massachusetts, USA). A cusp of aortic valves removed during surgery was immediately fixed in 10% neutral buffered formalin for 24h and then embedded in paraffin for histologic evaluations. Aortic valves leaflets preserved in RNAlater (ThermoFisher Scientific, Waltham, Massachusetts, USA) were disrupted and homogenized with the TissueRuptor® (Qiagen, Hilden, Germany). Total RNA was extracted with RNeasy® Fibrous Tissue Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Indexed libraries were prepared starting from 150 ng of purified RNA with TruSeq Stranded Total RNA Library Prep Kit (Illumina) according to the manufacturers instructions. Libraries size distribution was assessed using the Agilent 2200 TapeStation System (Agilent Technologies) and pooled in equimolar amounts, with a final concentration of 1,5 nM.