we reasoned that it would allow us to utilize all of our data while maintaining as much rigor in the analysis as possible.) Only microRNA-mRNA associations consistent with changes in microRNA proceeding changes in mRNA were considered. For example, microRNAs DE at 8h were paired with targets from the 8 and 120h time points but not the 0h time point. In all, six paired datasets were created for each brain region (S1 Table). Paired data sets are codified as TimePoint_DEmiR/TimePoint_DEtargets. For example, "0hDEmiR/120hDEtargets" specifies the microRNAs DE at 0h and their targets DE at 120h. We were particularly interested in genes dysregulated at 120h, which represents a state of extended withdrawal, as a result of microRNAs dysregulated at 0h (intoxication) and 8h (withdrawal). Thus, the datasets representing these temporal associations were evaluated with the "Core Analysis" option in IPA using minimum p value to resolve duplicate probes and Illumina MouseRef-8 version 2.0 as the reference set. Fig 1 provides a graphic overview of this method.
