Serum miRNA Profiling by NGS

The miRNA transcripts from serum samples were sequenced as described previously [18 (link)]. Briefly, miRNeasy Serum/Plasma Advanced Kit (Qiagen, Hilden, Germany) was used to purify total RNA from serum samples. RNA quantification was carried out using Qubit RNA Broad Range Assay Kit (Invitrogen, Carlsbad, CA, USA). The libraries were prepared using QIAseq miRNA next-generation sequencing (NGS) Library Kit (Qiagen) using QIAseq miRNA NGS 96 Index IL kit (Qiagen) for indexing. The quality-passed libraries, assessed using Qubit dsDNA HS assay kit (Invitrogen) and Agilent 2100 Bioanalyzer DNA1000 chip (Agilent Technologies, Santa Clara, CA, USA), were pooled using TruSeq PE Cluster Kit v3-cBot-HS kit (illumina, San Diego, CA, United States). HiSeq 3000/4000 SBS kit (illumina) was used to perform sequencing on illumina HiSeq 4000 system (10 million reads per sample).