Ion Torrent Sample Preparation, Sequencing and Analysis

Expression of 3,826 mouse genes was determined with a custom panel ordered from Thermo Fisher Scientific, use ion AmpliSeq library construction kit and run on the Ion Proton System for next generation sequencing according to manufacturer’s protocols (Thermo Fisher Scientific). 10 ng of mRNA for each of 6 samples were isolated using the miRcury mRNA Isolation Cell and Plant Kit (Exiqon) prior to reverse transcription with the Superscript VILO cDNA Sythesis Kit (Thermo Fisher Scientific). Data were analyzed using Torrent Suite Software 4.4 and Ion Reporter software (Thermo Fisher Scientific) and exported to Excel.

For normalization and downstream analysis, the edgeR package (27) was used to compute differential expression. The software’s built-in normalization methods and tagwise dispersion were used to determine the fold-change differential expression. Gene set enrichment analysis (GSEA) was performed on the differentially expressed genes using annotations from The Broad Institutes MSIG database (28) and correcting for false positives using the Benjamini-Hochberg method.