MicroRNA Sequencing and Analysis Protocol

MicroRNAs were sequenced using the NEBNext Small RNA library prep kit on an Illumina HiSeq 2000. The short reads were trimmed of adapters with Cutadapt [14 ]. Trimmed microRNA sequences greater than 17 nucleotides in length were then aligned to the reference genome and miRBase reference sequences using Bowtie [15 (link)]. Known microRNA expression and novel microRNA prediction and quantification were performed with miRDeep2 [16 (link)], using the CAP-miRSeq analysis pipeline [17 (link)]. Unsupervised hierarchical clustering was performed using the Pearson correlation method. ComiR, a computational tool for combinatorial microRNA target prediction was used to identify molecular pathways regulated by microRNAs that were differentially expressed between diseased and non-diseased palmar fascia [18 (link), 19 (link)]. The Database for Annotation and Visualization and Integrated Discovery v6.7 (DAVID 6.7) [20 (link), 21 (link)] was used to characterize functional gene clusters regulated by microRNA target genes.