Qiaseq mirna ngs library kit

Total RNA was purified from serum samples using miRNeasy Serum/Plasma Advanced Kit (Qiagen, Hilden, Germany) as per the manufacturer’s protocol. RNA concentrations were determined using Qubit RNA Broad Range Assay Kits (Invitrogen, CA, USA). Libraries were generated using QIAseq miRNA NGS Library Kit (Qiagen) according to manufacturer’s protocol. QIAseq miRNA NGS 96 Index IL kit (Qiagen) was used for indexing and the resulting libraries were quantified using Qubit dsDNA HS assay kit (Invitrogen) and its size distribution was determined using the Agilent 2100 Bioanalyzer DNA1000 chip (Agilent Technologies, Santa Clara, CA, USA). Quality-passed libraries were pooled, clustered using TruSeq PE Cluster Kit v3-cBot-HS (Illumina, San Diego, CA, USA) and sequenced using illumina HiSeq 4000 instrument at 10 million reads per sample utilizing HiSeq 3000/4000 SBS kit (Illumina) as per the manufacturer’s protocol.

Thawed samples were spun at ~2500 RPMs for five minutes to completely clear plasma of cells. Small RNAs were extracted from 500 μL plasma aliquots using the Exiqon (now Qiagen) miRCURY^TM RNA Biofluids Isolation Kit (Exiqon, Woburn, MA). Integrity, purity and quantity of purified miRNA was assessed using spectrophotometry and an Agilent 2100 Bioanalyzer capillary electrophoresis system (Agilent Technologies Inc, Palo Alto, CA). The Qiagen QIAseq miRNA NGS Library Kit was used for library preparation. MiRNAs were sequenced using an Illumina sequencer. Lab personnel were blinded to participant outcomes. Additional technical details are provided in Supplemental Materials.