Supporting Information Legends:
Table S1: Sample, RNA quality, gene counts, and library information. Sheet “Samples All ” lists all samples collected (sample ID and Admera Health ID for 3’ Tag-Seq and for the whole mRNA-Seq, NEB) with information about the treatment group they belong to, tissue type, sampling method, RIN value, and RNA concentration. Sample size used for each comparison and divided for tissue type, treatment group, and library preparation is also indicated. Sheet “3’ RNA Tag Seq ” lists all samples used for the 3’ Tag-Seq library with the following information for each sample: treatment group, sample ID, Admera Health ID’s, tissue type, sampling method, RIN value, concentration, raw read count, read count after mapping the randomly selected 11 million reads, and percentage of uniquely mapped genes on the reference genome. Sheet “Whole mRNA Seq ” lists all samples used in the whole mRNA-Seq library detailing for each sample the following: treatment group, sample ID, Admera Health ID’s (and new Admera Health ID if existing), tissue type, sampling method, RIN value, concentration, raw read count (PE and single), read count after mapping the randomly selected 40 million reads, and percentage of uniquely mapped genes on the reference genome. Sheet “Gene count ” lists all the 14 samples processed with both 3’ Tag-Seq and whole mRNA-Seq detailing for each sample the library type, IDs, gene count (using different number of reads per each gene as a filter), and percentage of uniquely mapped genes on the reference genome.
Table S2: Correlation coefficient values between samples for all comparisons. Comparisons among different treatment groups and tissues are indicated on separate sheets. Each sheet has an Admera Health sample ID, treatment group of belonging; depending on the comparisons information about library type (3’ Tag-Seq or whole mRNA-Seq NEB), sequencing platform, and tissue type are also provided.
Table S3: Output results of the Differential Expression Analysis . Results of Differential Expression Analysis done with DESeq2 for all comparisons, each of them presented on a separate sheet.
Table S4: Summary of gene expression patterns for different sampling methods and tissue types. The total numbers of genes with detectable expression for each sampling/tissue comparison are indicated along with the number and proportion of genes with significantly higher gene expression in one of the two tissues being compared for each sampling method.
Figure S1: Bar plot of transcript length versus number of non-expressed genes for each RNA-Seq library technique. In the legend next to the Figure, Full corresponds to whole mRNA-Seq and tag corresponds to 3’ Tag-Seq. Data based on the 14 samples sequenced using both techniques.
Figure S2: Sample-to-sample distance heatmap . Sample-to-sample distance heatmaps for the comparison between different sampling techniques and different tissue harvesting time for the different tissues. The rows and columns are arranged based on hierarchical clustering, so that samples with similar expression profiles are positioned near to each other. The color scale represents the distance between samples. A value of distance 0 indicates that two samples have identical gene expression. The smaller the distance is, the higher is the correlation between two samples. Treatment groups compared (called “condition ”) are indicated in different colors next to each heatmap. Condition 1 = fish captured by dip netting, Condition 2 = fish captured by electrofishing processed immediately, Condition 3 = fish captured by electrofishing processed 5 minutes after euthanasia.A. 3’ Tag-Seq dip netting versus electrofishing only for blood samples, B. 3’ Tag-Seq dip netting versus electrofishing only for gill samples, C. 3’ Tag-Seq dip netting versus electrofishing only for muscle samples, D. 3’ Tag-Seq electrofishing with immediate sampling versus electrofishing with delayed sampling only for blood samples, E. 3’ Tag-Seq electrofishing with immediate sampling versus electrofishing with delayed sampling only for gill samples, F. 3’ Tag-Seq electrofishing with immediate sampling versus electrofishing with delayed sampling only for muscle samples.
Figure S3: PCA plots showing PC1 and PC2 for samples that are differentially expressed among sampling techniques and tissue harvesting time for the different tissues. A. 3’ Tag-Seq dip netting versus electrofishing only for blood samples, B. 3’ Tag-Seq dip netting versus electrofishing only for gill samples, C. 3’ Tag-Seq dip netting versus electrofishing only for muscle samples,D. 3’ Tag-Seq electrofishing with immediate sampling versus electrofishing with delayed sampling only for blood samples, E.3’ Tag-Seq electrofishing with immediate sampling versus electrofishing with delayed sampling only for gill samples, F. 3’ Tag-Seq electrofishing with immediate sampling versus electrofishing with delayed sampling only for muscle samples.
Figure S4: Gene ontology (GO) heatmap based on over-representation analysis (ORA) of genes with greater tissue-specific expression. Rows represent GO descriptions and IDs for biological processes. Columns represent the sampling method [dip netting (N), electrofishing with rapid sampling (E), electrofishing with 5-minute wait time (E5)], followed by the tissue with significantly higher gene expression for listed GO terms, followed by the tissue with significantly lower gene expression. Heatmap intensity is a function of the false detection rate (FDR) of enriched GO terms.