The RNA samples used to generate the RNAseq data were also submitted to the Broad Institute’s Genomics Platform for processing on the Nanostring nCounter platform to generate miRNA profiles for 800 miRNAs using the Human V2 miRNA codeset. The complete list of miRNAs is available at https://www.nanostring.com/. 100 ng of each total RNA sample was used in the following Nanostring protocol: (1) multiplexed annealing of specific tags to their target miRNAs, (2) hybridization at 65 °C for 16 h, (3) enzymatic purification to remove unligated material, (4) scanning for 600 fields of view on the nCounter Digital Analyzer. Raw data were normalized using the internal positive spike-in controls and the average counts of all endogenous miRNAs in each lane to account for the variability in both the hybridization process and sample input. A metric yielding a detection call at a confidence level of 95% (P<0.05) was determined.
