Qiaseq mirna library kit

MiRNA library was prepared using QIAseq miRNA Library Kit (Qiagen). Before proceeding to cDNA conversion, NGS adaptor containing unique identifiers were ligated to the each 3′ and 5′ end of miRNA separately. Subsequently, NGS RT initiator, universal sequence and reverse transcription (RT) primer contains a combined unique molecular index (UMI) were added to the reaction, RT primer binds to a region of the 3′ adapter and enables conversion of the 3′/5′ ligated miRNA to cDNA. After RT, cDNA products were undergone cleanup procedure using a QIAseq miRNA NGS (QMN) beads. Next, library amplification was performed with HotStarTaq DNA Polymerase and wet universal forward primers that assigned with unique index (IDP). Finally, final products were cleaned with QMN bead. The reaction setup, components, concentrations and technical points were followed as per manufacturer’s instructions (QIAseq miRNA Library Kit Handbook for Illumina NGS Systems).

QIAseq miRNA Library Kit and Illumina NextSeq 500 platform were used for small RNA library preparation and sequencing, respectively, by the Ramaciotti Centre for Genomics (UNSW, Sydney, NSW, Australia).

RNA was extracted automatically from 500 μL of plasma using a Maxwell 48^® RSC Instrument together with the Maxwell^® RSC miRNA Plasma and Serum Kit (ref AS1680, Promega, USA) according to the manufacturer’s protocol. Libraries for small RNA sequencing were prepared using the QIAseq miRNA Library Kit for Illumina (Qiagen, Germany). The resulting small RNA libraries were concentrated by ethanol precipitation and quantified using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, USA) prior to sequencing on a Novaseq 6000 sequencer (Illumina, USA) with read lengths of 100 bases and 17 million single-end reads per sample, on average^{43 (link)–45 (link)}.

The Exiqon Genomics Services performed RNA extraction from the serum and small RNA sequencing (Hilden, Germany; n = 5). For 5 patients, 2 samples of 500 µL serum aliquot tubes (10 in total) were shipped to the Qiagen center (Hilden, Germany). The qPCR assay evaluated the quality of the tested samples. First, the expression levels of the samples were tested to see if the miRNA expressions were within the expected range for the miRNA content (hsa-miR-103a-3p, hsa-miR-191-5p, hsa-miR-451a, hsa-miR-23a-3p, and hsa-miR-30c-5p miRNAs are expressed in biofluids such as serum and plasma). The samples were then screened for the inhibition of enzymatic reactions (spike in control UniSp6) and potential hemolysis (miR23a-miR451a) [81 (link)]. The expression levels of the samples were within the expected range of miRNA content, and no inhibition or hemolysis was observed. The preparation of the small RNA library was then performed using the QIASeq miRNA library kit, including unique molecular identifiers (UMIs) for Illumina NGS systems (performed at the Qiagen center, Hilden, Germany). The single-end sequencing of 75 bp reads (50 bp in target and 25 bp for UMIs) was performed at a depth of 20 M, with one sample/lane in the Illumina Next-Seq 550.

For miR sequencing experiments, indexed libraries were prepared using 100 ng of total RNA as starting material, with a TruSeq Stranded Total RNA Sample Prep Kit and QIAseq miRNA Library Kit (Illumina Inc.), respectively. Libraries were sequenced (single‐end, 75 cycles) at a concentration of 8 pM/lane on the HiSeq 3000 platform (Illumina Inc.). Raw miRNA reads were preprocessed using FASTQC (Andrews, S. (2010) for quality control. Further, reads with Unique Molecular Identifiers (UMI) and low‐quality base calls were trimmed off using UMI‐tools²⁰ and Trim Galore,²¹ respectively. Processed reads were mapped to the reference mouse genome build (mm10) downloaded from UCSC (https://genome.ucsc.edu/index.html) using Bowtie.²² The R/Bioconductor package “DESeq2” was used to identify differentially expressed genes and miRs. Data were filtered according to read count value (threshold ≥6 reads). Only miRs having an adjusted P‐value of ≤0.05) and fold change value of 1.3 were considered for further analysis.