Sequencing coverage of each gene.

<p>The coverage of the protein-coding sequence of each gene of interest is tabulated, as a percentage, for a range of sequencing depths (≥1x, 10x, 20x, 30x, 50x and 100x reads).</p

Characteristics of six genes included in the assay.

<p>LRG: locus reference genomic (<a href="http://www.lrg-sequence.org" target="_blank">http://www.lrg-sequence.org</a>), LQT: Long QT syndrome, CPVT: catecholaminergic polymorphic ventricular tachycardia.</p

The 1 hour network cartoon built from the 89 genes differentially expressed at p<0.05.

<p>Genes differentially expressed to p<0.05 in human dermal microvascular EC treated for 1 hour with 10μM iron (II) citrate (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147990#pone.0147990.s009" target="_blank">S4 Table</a>) were entered into automated clustering programs, and clustered into 14 “biological process” annotation categories, with four additional terms obtained for “molecular function.” The network cartoon was built from the most inclusive and/or relevant representative terms from each cluster of gene ontology processes identified by the full set of 89 genes differentially expressed to p<0.05, and provides a schematic of processes operating differently in iron-treated EC compared to media treated EC. This approach resulted in connections between “iron binding,” through central positioned terms for DNA damage response/repair, programmed cell death (or apoptosis), cell cycle, and associated proteolysis through <i>SIAH1</i>. The top enrichment scoring term, vesicle mediated transport, finds itself at the lower part of the network linked to “response to wounding” through <i>LMAN1</i>.</p

Coverage of KCNQ1 and KCNH2 for the two platforms.

<p>Mean depth of coverage for 15 samples is shown for two genes on a log scale. Regions of no coverage therefore have negative values. The blue lines indicate local GC content (calculated with a 50 bp sliding window). Regions consistently missed have high GC content, with similar patterns for both platforms. KCNQ1 exons 1 & 8 and KCNH2 exons 1, 4 & 12 are difficult to sequence. A cartoon of the exon structure is shown beneath each panel. Plus (+) and minus (-) denote gene strand. Plots for all genes are shown in Supporting Information Figure S5. a.) MiSeq b.) Ion Torrent PGM.</p

Detection of coding variants for each NGS platform.

<p>Detection of coding variants for each NGS platform.</p

P53 protein expression.

<p><b>A)</b> Representative Western blot of p53 and GAPDH expression in HUVEC after treatment for 1 or 6 hours with fresh media or iron (II) citrate. (Original images supplied.) <b>B)</b> Quantifications of GAPDH (median and interquartile range displayed). <b>C)</b> P53 expression relative to GAPDH, p value calculated by Dunn’s test post Kruskal Wallis. Note in all four experiments, p53/GAPDH increased at 1 hour (minimum 1.5 fold; mean 2.1 [95% confidence intervals 1.8, 2.4] fold), and returned to baseline by 6 hours. <b>D)</b> P53/GAPDH protein changes in HUVEC treated with 40μM iron (II) citrate. Box plots demonstrate median, interquartile range, and two standard deviations.</p

Comparison of bench-top NGS platforms.

*<p>316 scale chip; ** average.</p

DNA damage response following iron treatments.

<p><b>A)</b> Representative images of DNA damage response (DDR) foci in HUVEC after treatment with 10μM iron (II) citrate, demonstrating To-Pro-3 nuclear staining (white, bottom left); p53-binding protein 1 (53BP1) staining (top left); punctate γH2AX foci (top right), and merged images (bottom right). <b>B)</b> Development of DDR foci in 10μM iron-treated endothelial cells. Box plots indicate median, interquartile range, and two standard deviations of the proportion of cells with DDR foci at the time points indicated after treatment with 10μM iron (II) citrate. Note the increase over the first hour, sustained after 24 hours. <b>C)</b> Comparison of DDR foci in endothelial cells treated with 10μM iron with and without media-rescue, and 40μM iron. Note the first hour increase was sustained after 24hr in either iron-treated, or media rescued cells, and was no greater in EC treated with 40μM iron for 1 hour.</p

Methodological validations of RNASeq in human microvascular endothelial cells.

<p><b>A)</b> Representative alignments from the four endothelial cell libraries described (HPMEC or HDMEC media (m) or iron (f)-treated), to pre-specified endothelial and non-endothelial specific micro(mi)RNAs and mRNAs. The 10 pre-specified miRNAs, and 10 pre-specified mRNAs, were either expressed strongly in endothelial cells, or recognized as non endothelial cell markers: the illustrated examples were representative of alignments to the other loci in the respective categories. Endothelial: miR-222, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147990#pone.0147990.ref023" target="_blank">23</a>] and VE-cadherin (CDH5, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147990#pone.0147990.ref024" target="_blank">24</a>], final exon illustrated). Non endothelial: miR-134, an example of a well known brain specific miRNA, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147990#pone.0147990.ref025" target="_blank">25</a>] and SM22α (TAGLN, smooth muscle actin, first exon). <b>B)</b> Representative coding strand exon alignments from all seven RNASeq libraries. Key: upper blocks with colored outlines: library-specific number of raw alignments, lower blue boxes: exons, lower black box: repeat element. Note the quasinormal alignments to the exons, and sharp exon/intron boundary definition. <b>C)</b> Linear regression and data plots of treatment-blinded miRNA alignments (log-transformed) and qt-PCR validations for all experiments performed with replicate treatment of replicate cells from identical donors. (Ct value is the cycle threshold when fluorescence exceeds background). For the 122 datasets from the same treatments in the same EC type but from different donors, the correlation r² was 0.22, p<0.0001 (data not shown).</p

qt-PCR Validations in endothelial cells.

<p><b>A)</b> qt-PCR of key transient early-rising mRNAs in human umbilical vein endothelial cells (HUVEC), selected due to relevant gene ontology processes: <i>LMAN1</i> encoding lectin, mannose-binding, 1 precursor implicated in tumorigenesis, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147990#pone.0147990.ref042" target="_blank">42</a>] and also involved in response to wounding, and ER-Golgi recycling; <i>SIAH1</i>, a tumor suppressor gene which encodes an E3 ubiquitin ligase that can induce apoptosis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147990#pone.0147990.ref043" target="_blank">43</a>]; and <i>RXRA</i> encoding retinoid X receptor alpha with key roles in cell proliferation, differentiation, and death[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147990#pone.0147990.ref044" target="_blank">44</a>]. <b>B)</b> qt-PCR validations in HUVEC of the key 6 hour genes implicated in DNA repair, <i>FANCG</i>, <i>BLM</i>, and <i>H2AFX</i>.</p