Qiaseq mirna library preparation kit

Small‐RNA Library preparations were executed with QIAseq miRNA Library Preparation Kit (1103679, Qiagen) according to the manufacturer's instructions using multiplexing adapters. Briefly, the small RNA fractions were first ligated to sequencing adapters from both 5′ and 3′ ends, reverse transcribed into cDNA using UMI‐assigning primers and purified using magnetic beads. A universal indexing sequence was also added in the reverse transcription step, thus allowing samples to be distinguished from each other. The samples were then amplified with standard thermocycler (Eppendorf), purified, and eluted into nuclease‐free water. Quality assessment of the libraries was completed with TapeStation 4200 (Agilent). The library sample concentrations were measured with Qubit fluorometer (Invitrogen), quantified, diluted, and pooled into a single mixture in equal amounts (1.8 pM per sample) prior to sequencing. Sequencing of the small‐RNA libraries were done with NextSeq 500 (Illumina) using NextSeq 500/550 High Output Kit v. 2.5 with 75 cycles (15057934, Illumina) to produce 75‐base pair single‐end reads with aimed mean sequencing depth of >5 M reads per sample as recommended by the manufacturer (Qiagen).

The small RNA libraries were prepared using the Qiaseq miRNA Library Preparation Kit (Qiagen, Hilden, Germany; catalogue no. 331505) following manufacturer’s instructions. Briefly, a universal preadenylated DNA adapter is added to the 3’ ends and an RNA adapter is ligated to the 5’ end. cDNA was synthesized using 3’-adapter as the primer. cDNA was subsequently cleaned up using magnetic QIAseq beads (provided with the library prep kit) and libraries were amplified and different 6-nt indices were included for each sample to enable sample identification after multiplexing. Library sizes were assessed using Bioanalyzer and average library sizes were around 180 bp. Samples were pooled and sequenced on Illumina Hiseq X sequencer generating 10 million single end reads per sample.