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.