Qiaseq mirna ngs 96 index il 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.

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).

For small RNA library preparation, 100 ng of RNA was subjected to 3' ligation followed by 5' ligation and reverse transcription. The next steps included QIAseq miRNA NGS (QMN) bead preparation, cDNA cleanup, and finally library amplification using tube indices, QIAseq miRNA NGS 96 Index IL kit (QIAGEN, Hilden, Germany). The yield of cDNA libraries was quantified using Qubit dsDNA HS assay kit (Invitrogen) and size distribution of the cDNA libraries were determined using the Agilent 2100 Bioanalyzer DNA1000 chip (Agilent Technologies). Libraries were subsequently pooled and were subjected to illumine sequencing at QBRI genomic core.