Differential expression of miRNAs during diapause
Differential expression analysis was done in R [v4.1.2 (R Core Team, 2021)] using the package DESeq2 [v3.17 (Love, Huber, & Anders, 2014)] on both the head and abdomen separately. Pairwise comparisons were made between expression at each timepoint to expression of miRNAs on diapause day 0, for both head and abdomen tissues. Differentially expressed miRNAs were then identified with a false discovery rate (FDR) of 0.05, and extracted for subsequent analyses. Global expression for differentially expressed miRNAs was visualized using multidimensional scaling of filtered read counts by applying the plotMDS function in the EdgeR package [v3.42.4 (Robinson, McCarthy, & Bioinformatics, 2020)].
Differentially expressed miRNAs were then clustered using fuzzy c-means clustering in the Mfuzz package [v3.17 (Kumar & Futschik, 2007)] separately for head and abdomen tissues. Normalized data were first standardized with a mean of zero and a standard deviation of one, then used to estimate the optimal fuzzifier using the mestimatefunction. The optimal numbers of clusters were determined based on minimum centroid distance (Kerr, Ruskin, Crane, & Doolan, 2008). A membership threshold of 0.8 was set for each cluster in each tissue for subsequent analyses.