Fig. 1

Whole transcriptome RNA-seq profile of larval zebrafish in response to low H₂O₂ treatment.,/p>

(a) Pools of ~500 larvae/set of 4 day-post-fertilized (dpf) zebrafish larvae were treated with 0.01% (3 mM) H₂O₂ for three hours and total RNA was subsequently collected followed by pair-end next generation RNA sequencing (n = 3 biological replicates). (b) H₂O₂ sensor (HPF) either alone or with H₂O₂ treatment shows that H₂O₂ is mostly retained in the skin epithelium (n = 5 fish). (c) Distribution of mapped reads in the zebrafish transcriptome. RNA-seq data sent to NCBI (GEO: GSE75728). (d) Transcript complexity between untreated and H₂O₂-treated larval zebrafish samples. Based on read CPM (counts per million), the left-most value on the X-axis represents the most highly expressed transcripts, which is incrementally summed with each successively lower expressed transcript (rightward). The y-axis (% contribution of the total transcripts) was calculated using: [CPM/sum of all CPM] x 100%. (e) RNA-seq data was normalized with the read CPM method of the number of mapped reads on gene exons. Transcript expression data transformed on M (log ratio of fold change) and A (mean average) scale. Boxed blue regions represent statistically significant transcripts (p < 0.05) returned by the test for differential expression. The MA-plot shows the log₂ fold changes from the treatment over the mean of normalized counts, i.e. the average of counts normalized by size factor. Cutoff set to >1 log₂ CPM averaged over all samples, and below the cutoff there is no real inferential power. Note: Statistical significance drops below the threshold. (f) Quantitative PCR validation of RNA-seq results of a sub-set of candidate targets. Full data set is presented in Table S7 (n = 3 biological replicates). (g) Heat map indicates unsupervised hierarchical clustering of the top (left) and bottom (right) most significantly enriched transcripts derived from the RNA-seq data after H₂O₂ treatment. Hierarchical clustering was performed between individual experiments and transcripts. The color key indicates the log₂ CPM expression values. Abbreviations: HPF (hydrogen peroxide fluorogenic probe or pentafluorobenzenesulfonyl-fluorescein), CPM: Counts per million, FC: Fold-change.