alias: GSM4544820
title: GSM4544820: Leaflet sample V15; Homo sapiens; RNA-Seq
library_construction_protocol: Conduits for NGS were explanted from the patients during surgical procedures and immediately transferred to RNAlater® (Sigma Aldrich) to inhibit RNA-degradation. Explants were then dissected to isolate the thickened material of the conduit-wall and one of the three valve-leaflets for RNA-isolation. RNA-isolation was performed in an RNase free area using the TRIzol® reagent (ThermoFisher, 15596026) according to manufacturer's recommendations. Briefly, small tissue-pieces were frozen in liquid nitrogen and then powered using the TissueLyser® (Qiagen). The resulting 50 – 100 mg tissue-powder was lysed in 1mL TRIzol reagent. Then, the lysate was mixed with 0,2 mL Chloroform and centrifuged to create three phases with the RNA accumulating in the upper aqueous phase. RNA was precipitated using isopropylalcohol and washed twice using 75% Ethanol. DNase treatment was performed on RNeasy mini-columns according to the manufacturer's protocols (Qiagen). RNA quality was evaluated using the NanoDrop (ThermoFisher) and a fragment analyzer. RNA-seq libraries were performed using 500 ng total RNA of a non stranded RNA Seq, massively-parallel mRNA sequencing approach from Illumina (TruSeq stranded total RNA Library Preparation , Illumina)
identifiers.namespace: GEO
identifiers.id: GSM4544820