Differential Expression (DE)
We created a gene-level read count matrix for all samples usingfeatureCounts (Liao et al., 2014). The read count matrix was
filtered for a minimum count cutoff = 3 cpm over at least two replicates
per comparable group. All DE analyses were performed using the R packageedgeR (Robinson et al., 2009) after TMM library normalization.
Normalized read counts were analyzed by Generalized Linear Models (GLM)
assuming a negative binomial model of read counts. All comparisons were
performed using a full factorial design that included parental and
offspring acclimation treatments using three biological replicates.
Transcriptional TGP was assessed by comparing samples whose parents were
acclimated at 36 °C vs 25 °C, while WGP was detected by comparing
relative expression changes between offspring samples acclimated at 36
°C vs 25 °C. Genes with a false-discovery rate (FDR) corrected p-value
of < 0.05 (Benjamini and Hochberg, 1995) and a
log2-fold-change threshold of > 1.0 were
considered significant. A correlation matrix comparing relative
expression levels of significant DE genes was then generated in order to
investigate the relationship between transcriptional WGP and TGP.