RNA-seq Analysis of miRNA Transcriptome

RNA integrity and absence of DNA was confirmed by Bioanalyzer RNA Nano chips (Agilent Technologies, St. Louis, MO) and Qubit DNA High sensitivity kit, respectively. Sequencing libraries were prepared using the Illumina TruSeq Small RNA Library Preparation Kit. Next generation sequencing was performed on 2 lanes of an Illumina HiSeq2500 (Illumina, San Diego, CA) multiplexing all samples (single end read, 50 bp). The quality of sequencing reads was verified using FastQC0.11.5 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) before and after trimming. Adapters were trimmed using cutadapt (18 (link)). Reads with <15 bp and >40 bp insert sequences were discarded. Alignment of reads was performed using miRBase V21 (19 (link)) with sRNAbench (20 (link)). For normalization and identification of differentially expressed miRNAs EdgeR and DeSeq in R was used (21 (link)–23 (link)). miRNAs with an at least 5 raw count per library were included. Disease groups were compared using the unpaired Mann–Whitney test, and, to decrease the false discovery rate, a corrected p-value was calculated using the Benjamini–Hochberg method. Adjusted p < 0.05 and log2 fold of change >1.5 were the cut-off for significance. The RNA seq data generated in this study have been submitted to the NCBI GEO with accession number GSE156693.