4.12. Small RNAseq from Fresh Frozen Tissues
In total, 24 RNA samples from 19 patients were sent for small RNAseq profiling. Total RNA samples were size-selected for miRNA, and libraries were constructed using NEBNext adaptors, followed by single-read 50 sequencing on the Ilumina HiSeq 4000. Reads were trimmed using Trimmomatic (v0.35) (Bolger, AM 2014 2114), removing adaptors and low-quality bases at the end of each read using a 4 bp sliding window to trim where average window quality fell under 30. Trimmed reads <15 bp were discarded. Clean reads were aligned using novoalign (Novocraft). Small RNA expression levels were obtained using featureCount on the hg19 miRbase annotation. Differential expression analysis was performed using DESeq2. Small RNAs were considered significantly differentially expressed if they met the following criteria: average expression across samples ≥ 50, |log2 Fold Change| ≥ 1, and FDR ≤ 0.05.