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.