miRNA Expression Profiling by Small RNA-Seq

Initial small-RNA libraries were prepared from 1 μg total RNA (TruSeq Small RNA kit, Illumina), followed by miRNA enrichment (Caliper LabChipXT, PerkinElmer) according to manufacturer’s protocols. Small RNA-Seq libraries were sequenced on Illumina HiSeq 2000. Library preparation and sequencing were conducted in FIMM Sequencing Laboratory, University of Helsinki, Finland. Quality control of the raw reads was performed using FastQC (ver. 0.11.7) and MultiQC (ver. 1.7) (Ewels et al., 2016 (link)). Trimmomatic (ver. 0.38) was implemented to remove adapters and trim the quality of reads with the following settings - ILLUMINACLIP:2:30:9, LEADING:3, CROP:50, TRAILING:3, SLIDINGWINDOW:4:20, MINLEN:16. Reads were aligned to human genome reference GRCh38 using bowtie (ver. 1.2.2, settings: -n 1 -l 20 -q -m 40 -k 1 -t –best –strata) (Langmead et al., 2009 (link)). miRNA quantification was performed using featureCounts from the Rsubread package (ver. 1.20.6) (Liao et al., 2019 (link)) for R with miRNA annotations from miRBase 22.1 as reference (Kozomara et al., 2019 (link)).