GSM6482227: HUVEC_LSS_rep1; Homo sapiens; RNA-Seq
Cellular RNA was isolated using the NucleoSpin RNA XS isolation kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's instructions. After initial quality control using Agilent's Bioanalyzer, sequencing libraries were prepared from 500 ng of total RNA per sample following Roche's stranded “KAPA RNA HyperPrep” library preparation protocol for single indexed Illumina libraries: First, the polyA-RNA fraction was enriched using oligo-dT-probed paramagnetic beads. Enriched RNA was heat-fragmented and subjected to first strand synthesis using random priming. The second strand was synthesized incorporating dUTP instead of dTTP to preserve strand information. Afterwards, A-tailing Illumina sequencing compatible adapters were ligated. Following bead-based clean-up steps, the libraries were amplified using 10 cycles of PCR. Library quality and size was checked with qBit, Agilent Bioanalyzer, and qPCR. Sequencing was carried out in biological triplicates on an Illumina HiSeq 4000 system in SR75bp mode (single read, 75 bp read length) yielding between 46 and 69 million fragments per sample.