Serum miRNA Profiling by RNA-Seq

RNA-Seq was performed on collected samples as previously described (41 (link)). Briefly, circulating miRNA from serum samples (200 μl) were extracted using miRNeasy Serum/Plasma Advanced Kit (Qiagen, Hilden, Germany) and RNA concentrations were measured by Qubit RNA Broad Range Assay Kit (Invitrogen, CA, USA). Library preparation was carried out using QIAseq miRNA NGS Library Kit (Qiagen) and indexing was done using QIAseq miRNA NGS 96 Index IL kit (Qiagen). The quality control measures for generated libraries were performed using Qubit dsDNA HS assay kit (Invitrogen) and Agilent 2100 Bioanalyzer DNA1000 chip (Agilent Technologies, Santa Clara, CA, USA). The pooled libraries were clustered using TruSeq PE Cluster Kit v3-cBot-HS (illumina, San Diego, CA, USA). Sequencing was performed on illumina HiSeq 4000 system (10 million reads per sample) using HiSeq 3000/4000 SBS kit (illumina).