RNA sequencing of human pulmonary artery endothelial cells transfected with miR-181 and miR-324

Pulmonary arterial hypertension (PAH) is a severe disorder of lung vasculature that causes right heart failure. Dysregulation of several microRNAs (miRNAs), small (~22 nucleotide long) non-coding RNAs that negatively regulate gene expression, has been demonstrated in human and animal PAH. miR-181a-5p and miR-324-5p attenuated apoptosis, inflammatory activation and proliferation of Human Pulmonary Artery Endothelial Cells (HPAECs).To identify targets of potential therapeutic significance, HPAECs were transfected with control miRNA (non-targeting transfection control; Ambion Life Technologies, 4464076) at 20 nmol/L or control miRNA at 10 nmol/L plus either miR-181a-5p mimic (10 nmol/L; Ambion Life Technologies; miRVanaTM miRNA mimic, 4464066, ID MC10421) or miR-324-5p (10 nmol/L; miRVanaTM miRNA mimic, 4464066, ID MC10253) with Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher, 13778150). RNA was extracted from trypsinised HPAECs using miRCURY RNA Isolation Kit (Exiqon), according to manufacturer’s instructions.Next-generation RNA-sequencing of transfected HPAECs was performed in duplicate at the Imperial BRC Genomics Facility (Imperial College of London, UK). RNA libraries were prepared using TruSeq® Stranded mRNA HT Sample Prep Kit (Illumina Inc.) according to the manufacturer’s protocol. Briefly, 1 μg of high-quality total RNA (RNA Integrity Number Score ≥ 8.0) was used for polyadenylated RNA selection using poly-T oligo attached magnetic beads, followed by the fragmentation of poly-A containing mRNA. Cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase with random primers. The cDNA was further converted into double-stranded DNA that was end-repaired to incorporate the specific index adapters for multiplexing, followed by purification and amplification. The amplified libraries were examined using an Agilent 2100 Bioanalyzer and a Qubit.The samples for this study were pooled alongside 12 additional samples from other studies and together the 18 samples were sequenced across 4 lanes (2 x 100 bp) on a HiSeq 2500 using TruSeq SBS V3-HS kit (Illumina Inc.) in high output run mode (average of 34.4 million reads per sample).