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            <gmx:Anchor xlink:href="http://lod.bco-dmo.org/id/dataset/1004689.rdf" xlink:actuate="onRequest">KEGG gene differential expression data for mixotrophs and diatoms from an iron incubation experiment during the PUPCYCLE II R/V Sally Ride cruise within the California Current System in May-June 2023</gmx:Anchor>
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            <gco:CharacterString>Cite this dataset as: Speciale, E., Lim, P., Jeong, Y., Cook, C., Cohen, N., McClure, W., Schnetzer, A., Marchetti, A. (2026) KEGG gene differential expression data for mixotrophs and diatoms from an iron incubation experiment during the PUPCYCLE II R/V Sally Ride cruise within the California Current System in May-June 2023. Biological and Chemical Oceanography Data Management Office (BCO-DMO). (Version 1) Version Date 2026-09-03 [if applicable, indicate subset used]. doi:10.26008/1912/bco-dmo.1004689.1 [access date]</gco:CharacterString>
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        <gco:CharacterString>KEGG gene differential expression from PUPCYCLE II Dataset Description: &amp;lt;p&amp;gt;This dataset is one of many generated from an iron incubation experiment conducted as part of the PUPCYCLE II cruise in May and June 2023. The different analyses are listed here, with links to other datasets from this study in the Related Datasets section.&amp;lt;/p&amp;gt;

&amp;lt;p&amp;gt;Analyses&amp;amp;nbsp;include:&amp;lt;/p&amp;gt;

&amp;lt;ul&amp;gt;
&amp;lt;li&amp;gt;Physiology (dataset 994607)
&amp;lt;ul&amp;gt;
&amp;lt;li&amp;gt;Dissolved inorganic nutrients (nitrate, phosphate, silicic acid)&amp;lt;/li&amp;gt;
&amp;lt;li&amp;gt;Size-fractionated chlorophyll a&amp;lt;/li&amp;gt;
&amp;lt;li&amp;gt;Size-fractionated isotope uptake rates (nitrate and DIC)&amp;lt;/li&amp;gt;
&amp;lt;li&amp;gt;Flow cytometry&amp;lt;/li&amp;gt;
&amp;lt;li&amp;gt;FlowCAM analysis&amp;lt;/li&amp;gt;
&amp;lt;li&amp;gt;FLP experiments for mixotroph cell-specific grazing rates&amp;lt;/li&amp;gt;
&amp;lt;/ul&amp;gt;
&amp;lt;/li&amp;gt;
&amp;lt;li&amp;gt;RNA taxonomy data for normalized reads (dataset 1003525)&amp;lt;/li&amp;gt;
&amp;lt;li&amp;gt;Differential expression for +Fe versus DFB treatments at T1 and T2: Pfam
&amp;lt;ul&amp;gt;
&amp;lt;li&amp;gt;Mixotroph Pfam (dataset 1004659)&amp;lt;/li&amp;gt;
&amp;lt;li&amp;gt;Diatom Pfam (dataset 1004674)&amp;lt;/li&amp;gt;
&amp;lt;/ul&amp;gt;
&amp;lt;/li&amp;gt;
&amp;lt;li&amp;gt;&amp;lt;strong&amp;gt;Differential expression for +Fe versus DFB treatments at T1 and T2 for mixotrophs and diatoms: KEGG gene&amp;amp;nbsp;(this dataset 1004689)&amp;lt;/strong&amp;gt;&amp;lt;/li&amp;gt;
&amp;lt;li&amp;gt;Differential expression for +Fe versus DFB treatments at T1 and T2 for mixotrophs and diatoms: KEGG pathways (dataset 1004704)&amp;lt;/li&amp;gt;
&amp;lt;li&amp;gt;Dissolved iron concentrations (dataset 1004007)&amp;lt;/li&amp;gt;
&amp;lt;/ul&amp;gt;

&amp;lt;p&amp;gt;See Related Datasets section below for links to above mentioned datasets.&amp;lt;/p&amp;gt; Methods and Sampling: &amp;lt;p&amp;gt;Data collection took place on the R/V Sally Ride from May 29th to June 10th, 2023. To simulate upwelling conditions under different iron treatments, an onboard incubation experiment was conducted. Seawater for the incubation experiment was collected within the northern CCS, off the southern coast of Oregon at 43°02'42.7&amp;quot;N, 124°33'07.2&amp;quot;W. The collected seawater was deemed as freshly upwelled water and taken from a depth of 55 meters – which corresponds to a depth slightly below the euphotic zone receiving less than 1% irradiance – using trace-metal clean techniques on May 30th, 2023, 13:30 GMT. The seawater was pumped and homogenized using trace metal clean techniques, then transferred into a total of thirty 20 L low-density polyethylene cubitainers. Three cubitainers were immediately harvested for the initial timepoint (T0). The remaining twenty-seven cubitainers were assigned treatments: nine were unamended (Ctrl), nine were amended with 5 nM FeCl&amp;lt;sub&amp;gt;2&amp;amp;nbsp;&amp;lt;/sub&amp;gt;(+Fe), and nine were amended with 200 nM desferrioxamine B, a strong iron chelator that inhibits dissolved iron uptake (DFB). The cubitainers were placed in an on-deck incubator covered with two layers of neutral density screening to simulate 26% of incident&amp;amp;nbsp;irradiance. The incubator was&amp;amp;nbsp;supplied with flow-through surface seawater to maintain ambient surface temperature. Three cubitainers from each treatment were harvested for each of the three subsequent timepoints: 48 hours (T1), 168 hours (T2), and 264 hours (T3) after incubation.&amp;lt;/p&amp;gt;

&amp;lt;p&amp;gt;&amp;lt;strong&amp;gt;RNA Collection and Metatranscriptomics.&amp;amp;nbsp;&amp;lt;/strong&amp;gt;Cubitainers were harvested for RNA by collecting and filtering approximately 2.5 to 4 L of each sample onto 0.8 μm Pall Supor filters (142 mm) using a peristaltic pump; samples were then immediately flash frozen in liquid nitrogen and stored at -80°C. RNA was extracted using the RNAqueous-4PCR kit, according to the manufacturer's instructions with a few modifications – filters were cut up due to their large size, 200 μL of glass beads were added, and 3 mL of lysis buffer was added. RNA samples were sent to GENEWIZ for library preparation and sequencing with PolyA tail selection. Sequencing was performed on an Illumina HiSeq 4000 with a 2x150 bp configuration. GENEWIZ provided raw paired-end read sequences for each sample.&amp;amp;nbsp;&amp;lt;/p&amp;gt;

&amp;lt;p&amp;gt;Raw reads were trimmed using Trim Galore v0.6.10 (Krueger, 2015), which uses Cutadapt (Martin, 2011), and quality control was assessed through FastQC (Andrews, 2010). A&amp;amp;nbsp;&amp;lt;em&amp;gt;de novo&amp;amp;nbsp;&amp;lt;/em&amp;gt;metatranscriptome assembly was conducted using rnaSPAdes v3.15.5 for individual assemblies (Bushmanova et al., 2019) and CD-HIT v4.8.1 for a grand assembly (Li &amp;amp;amp; Godzik, 2006). TransDecoder v5.7.1 was used to determine the most likely open reading frame (ORF) for each contig (Haas, 2023). ORFs were annotated using the Marine Functional Eukaryotic Reference Taxa (MarFERReT) database v1.1.1 (e-value &amp;amp;lt; 1e&amp;lt;sup&amp;gt;-06&amp;lt;/sup&amp;gt;) (Groussman et al., 2023) for NCBI taxonomies (Federhen, 2011), PR&amp;lt;sup&amp;gt;2&amp;lt;/sup&amp;gt;&amp;amp;nbsp;taxonomies (Guillou et al., 2012) and Pfam 34.0 functions (Mistry et al., 2021). To remove any potential bacterial contamination, ORFs were also annotated against the PhyloDB v1076 database using EUKulele (e-value &amp;amp;lt; 1e&amp;lt;sup&amp;gt;-06&amp;lt;/sup&amp;gt;) (Krinos et al., 2020). Any ORF with a higher bitscore for a bacteria or virus compared to its MarFERReT annotation was removed (resulting in &amp;amp;lt; 3% of ORFs removed). To maximize functional annotation coverage, ORFs were annotated using eggNOG-mapper v2.1.12 (Cantalapiedra et al., 2021) to obtain annotations from the Kyoto Encyclopedia of Genes and Genomes (KEGG) (Kanehisa, 2000). Trimmed samples were then aligned to the grand assembly using Salmon v1.10.3 (Patro et al., 2017) and exported into a comprehensive counts table using tximport (Soneson et al., 2016).&amp;amp;nbsp;Reads not mapping to a protist group as defined by MarFERReT annotations were removed before downstream analysis.&amp;amp;nbsp;&amp;lt;/p&amp;gt;

&amp;lt;p&amp;gt;DESeq2 was used to conduct differential expression analyses (Love et al., 2014). Differential expression analyses for KEGG genes were performed for reads mapping to mixotroph genera and reads mapping to the lineage Bacillariophyta (henceforth referred to as diatoms). Reads not mapping to the taxa group being analyzed were removed prior to normalization and statistical testing. Mixotrophs were identified based on constitutive mixotroph genera within the Mixoplankton Database (MDB) (Mitra et al., 2023 ). Reads were summed based on annotation ID prior to entering DESeq2 analysis; if a contig annotated to multiple IDs, its reads were evenly distributed between the mapped annotations. The differential expression analyses only focused on comparing +Fe vs DFB at T1 and T2.&amp;amp;nbsp;&amp;lt;/p&amp;gt;

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        <gmx:Anchor xlink:href="http://lod.bco-dmo.org/id/award/768005.rdf" xlink:title="OCE-1751805" xlink:actuate="onRequest">Funding provided by NSF Division of Ocean Sciences (NSF OCE) Award Number: OCE-1751805 Award URL: https://www.nsf.gov/awardsearch/show-award?AWD_ID=1751805</gmx:Anchor>
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	Units: decimal degrees
	Description: &lt;p&gt;Latitude of water sampling site&lt;/p&gt; 
http://lod.bco-dmo.org/id/dataset-parameter/1006975.rdf
	Name: Longitude
	Units: decimal degrees
	Description: &lt;p&gt;Longitude of water sampling site&lt;/p&gt; 
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                <gco:CharacterString>&amp;lt;p&amp;gt;Data collection took place on the R/V Sally Ride from May 29th to June 10th, 2023. To simulate upwelling conditions under different iron treatments, an onboard incubation experiment was conducted. Seawater for the incubation experiment was collected within the northern CCS, off the southern coast of Oregon at 43°02'42.7&amp;quot;N, 124°33'07.2&amp;quot;W. The collected seawater was deemed as freshly upwelled water and taken from a depth of 55 meters – which corresponds to a depth slightly below the euphotic zone receiving less than 1% irradiance – using trace-metal clean techniques on May 30th, 2023, 13:30 GMT. The seawater was pumped and homogenized using trace metal clean techniques, then transferred into a total of thirty 20 L low-density polyethylene cubitainers. Three cubitainers were immediately harvested for the initial timepoint (T0). The remaining twenty-seven cubitainers were assigned treatments: nine were unamended (Ctrl), nine were amended with 5 nM FeCl&amp;lt;sub&amp;gt;2&amp;amp;nbsp;&amp;lt;/sub&amp;gt;(+Fe), and nine were amended with 200 nM desferrioxamine B, a strong iron chelator that inhibits dissolved iron uptake (DFB). The cubitainers were placed in an on-deck incubator covered with two layers of neutral density screening to simulate 26% of incident&amp;amp;nbsp;irradiance. The incubator was&amp;amp;nbsp;supplied with flow-through surface seawater to maintain ambient surface temperature. Three cubitainers from each treatment were harvested for each of the three subsequent timepoints: 48 hours (T1), 168 hours (T2), and 264 hours (T3) after incubation.&amp;lt;/p&amp;gt;

&amp;lt;p&amp;gt;&amp;lt;strong&amp;gt;RNA Collection and Metatranscriptomics.&amp;amp;nbsp;&amp;lt;/strong&amp;gt;Cubitainers were harvested for RNA by collecting and filtering approximately 2.5 to 4 L of each sample onto 0.8 μm Pall Supor filters (142 mm) using a peristaltic pump; samples were then immediately flash frozen in liquid nitrogen and stored at -80°C. RNA was extracted using the RNAqueous-4PCR kit, according to the manufacturer's instructions with a few modifications – filters were cut up due to their large size, 200 μL of glass beads were added, and 3 mL of lysis buffer was added. RNA samples were sent to GENEWIZ for library preparation and sequencing with PolyA tail selection. Sequencing was performed on an Illumina HiSeq 4000 with a 2x150 bp configuration. GENEWIZ provided raw paired-end read sequences for each sample.&amp;amp;nbsp;&amp;lt;/p&amp;gt;

&amp;lt;p&amp;gt;Raw reads were trimmed using Trim Galore v0.6.10 (Krueger, 2015), which uses Cutadapt (Martin, 2011), and quality control was assessed through FastQC (Andrews, 2010). A&amp;amp;nbsp;&amp;lt;em&amp;gt;de novo&amp;amp;nbsp;&amp;lt;/em&amp;gt;metatranscriptome assembly was conducted using rnaSPAdes v3.15.5 for individual assemblies (Bushmanova et al., 2019) and CD-HIT v4.8.1 for a grand assembly (Li &amp;amp;amp; Godzik, 2006). TransDecoder v5.7.1 was used to determine the most likely open reading frame (ORF) for each contig (Haas, 2023). ORFs were annotated using the Marine Functional Eukaryotic Reference Taxa (MarFERReT) database v1.1.1 (e-value &amp;amp;lt; 1e&amp;lt;sup&amp;gt;-06&amp;lt;/sup&amp;gt;) (Groussman et al., 2023) for NCBI taxonomies (Federhen, 2011), PR&amp;lt;sup&amp;gt;2&amp;lt;/sup&amp;gt;&amp;amp;nbsp;taxonomies (Guillou et al., 2012) and Pfam 34.0 functions (Mistry et al., 2021). To remove any potential bacterial contamination, ORFs were also annotated against the PhyloDB v1076 database using EUKulele (e-value &amp;amp;lt; 1e&amp;lt;sup&amp;gt;-06&amp;lt;/sup&amp;gt;) (Krinos et al., 2020). Any ORF with a higher bitscore for a bacteria or virus compared to its MarFERReT annotation was removed (resulting in &amp;amp;lt; 3% of ORFs removed). To maximize functional annotation coverage, ORFs were annotated using eggNOG-mapper v2.1.12 (Cantalapiedra et al., 2021) to obtain annotations from the Kyoto Encyclopedia of Genes and Genomes (KEGG) (Kanehisa, 2000). Trimmed samples were then aligned to the grand assembly using Salmon v1.10.3 (Patro et al., 2017) and exported into a comprehensive counts table using tximport (Soneson et al., 2016).&amp;amp;nbsp;Reads not mapping to a protist group as defined by MarFERReT annotations were removed before downstream analysis.&amp;amp;nbsp;&amp;lt;/p&amp;gt;

&amp;lt;p&amp;gt;DESeq2 was used to conduct differential expression analyses (Love et al., 2014). Differential expression analyses for KEGG genes were performed for reads mapping to mixotroph genera and reads mapping to the lineage Bacillariophyta (henceforth referred to as diatoms). Reads not mapping to the taxa group being analyzed were removed prior to normalization and statistical testing. Mixotrophs were identified based on constitutive mixotroph genera within the Mixoplankton Database (MDB) (Mitra et al., 2023 ). Reads were summed based on annotation ID prior to entering DESeq2 analysis; if a contig annotated to multiple IDs, its reads were evenly distributed between the mapped annotations. The differential expression analyses only focused on comparing +Fe vs DFB at T1 and T2.&amp;amp;nbsp;&amp;lt;/p&amp;gt;

&amp;lt;p&amp;gt;&amp;amp;nbsp;&amp;lt;/p&amp;gt;</gco:CharacterString>
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                <gco:CharacterString>&amp;lt;p&amp;gt;Differential expression analysis was conducted through DESeq2’s “DESeq()” function in which significance is determined by estimating size factors (using median-of-ratios method), estimating dispersion, fitting a negative binomial generalized linear model, then hypothesis testing. For hypothesis testing, the Wald test is used followed by the Benjamini-Hochberg procedure for false discovery rate (which gives the p-adjusted value). A pathway was considered differentially expressed if the absolute value of log2FoldChange &amp;amp;gt; 1 and p-adjusted &amp;amp;lt; 0.05.&amp;lt;/p&amp;gt;

&amp;lt;p&amp;gt;The metatranscriptomic pipeline code used for this project is publicly available at https://github.com/emilyspeciale/Speciale-Metatranscriptomics.&amp;amp;nbsp;&amp;lt;/p&amp;gt;

&amp;lt;p&amp;gt;&amp;lt;strong&amp;gt;Software:&amp;lt;/strong&amp;gt;&amp;amp;nbsp;Trim Galore v0.6.10; FastQC&amp;amp;nbsp;v0.12.1; rnaSPAdes v3.15.5; CD-HIT v4.8.1; TransDecoder v5.7.1; EUKulele v2.0.9; eggNOG-mapper v2.1.12; Salmon v1.10.3; &amp;amp;nbsp;tximport v1.3.9; DESeq2.&amp;lt;/p&amp;gt;

&amp;lt;p&amp;gt;&amp;lt;strong&amp;gt;Databases/reference resources:&amp;lt;/strong&amp;gt;&amp;amp;nbsp;MarFERReT v1.1.1; NCBI Taxonomy; PR2; Pfam 34.0; PhyloDB v1076;&amp;amp;nbsp;&amp;lt;span style=&amp;quot;font-family:arial,sans-serif; font-size:13.3333px&amp;quot;&amp;gt;Kyoto Encyclopedia of Genes and Genomes (KEGG)&amp;lt;/span&amp;gt;; Mixoplankton Database (MDB).&amp;lt;/p&amp;gt;

&amp;lt;ul&amp;gt;
&amp;lt;li&amp;gt;1. Raw reads were trimmed with Trim Galore v0.6.10 (Krueger, 2015)&amp;lt;/li&amp;gt;
&amp;lt;li&amp;gt;2. Read quality was assessed with FastQC v0.12.1 (Andrews, 2010)&amp;lt;/li&amp;gt;
&amp;lt;li&amp;gt;3.&amp;amp;nbsp;De novo metatranscriptome assemblies were generated&amp;amp;nbsp;using rnaSPAdes v3.15.5 (Bushmanova et al., 2019) for individual assemblies and&amp;amp;nbsp;CD-HIT v4.8.1 (Li &amp;amp;amp; Godzik, 2006) for the grand assembly&amp;lt;/li&amp;gt;
&amp;lt;li&amp;gt;4. ORFs were identified with&amp;amp;nbsp;TransDecoder v5.7.1 (Haas, 2023).&amp;amp;nbsp;&amp;lt;/li&amp;gt;
&amp;lt;li&amp;gt;5. Taxonomy and functional annotation via Marine&amp;amp;nbsp;Functional Eukaryotic Reference Taxa (MarFERReT) database v1.1.1 (Groussman et al., 2023); includes&amp;amp;nbsp;NCBI taxonomies (Federhen, 2011),&amp;amp;nbsp;PR2 taxonomies (Guillou et al., 2012), and&amp;amp;nbsp;Pfam 34.0 functions (Mistry et al., 2021).&amp;amp;nbsp;&amp;lt;/li&amp;gt;
&amp;lt;li&amp;gt;6. PhyloDB v1076 filtering using EUKulele v2.0.9 (Krinos et al., 2020).&amp;amp;nbsp;&amp;lt;/li&amp;gt;
&amp;lt;li&amp;gt;7. eggNOG-mapper v2.1.12 (Cantalapiedra et al., 2021) /&amp;amp;nbsp;KEGG annotation (Kanehisa, 2000)&amp;lt;/li&amp;gt;
&amp;lt;li&amp;gt;8. Reads were quantified with Salmon v1.10.3 (Patro et al., 2017)&amp;amp;nbsp;&amp;lt;/li&amp;gt;
&amp;lt;li&amp;gt;9. Reads were imported with tximport v1.3.9 (Soneson et al., 2016)&amp;lt;/li&amp;gt;
&amp;lt;li&amp;gt;10. Taxonomic filtering where reads outside the target taxonomic group were removed&amp;lt;/li&amp;gt;
&amp;lt;li&amp;gt;11. Annotation-ID read summation&amp;lt;/li&amp;gt;
&amp;lt;li&amp;gt;12. DESeq2 v1.42.1 (Love et al., 2014) was used to compare +Fe versus DFB at T1 and T2&amp;lt;/li&amp;gt;
&amp;lt;/ul&amp;gt;

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                  <gco:CharacterString>CURATION ACTIONS PERFORMED ON DATA 
- Loaded source file &amp;quot; BCO_DMO_PUPCYCLEII_KEGGgeneDE.csv&amp;quot; into the BCO-DMO system converting missing data identifier NA to empty cell.
- Added columns for latitude and longitude of water sampling station
- Set data types for first 6 columns to string, and all others to numeric, with p-values in scientific notation.
- Exported the final file as &amp;quot;1004689_v1_kegg_genes_diffexp.csv&amp;quot;

CURATION ACTIONS PERFORMED ON METADATA
- Added summary for broader study showing connections between related datasets
- Changed in-line citations to have format of (author, year) replacing number references.
- Corrected the FastQC citation to Andrews et al. (2010)

ISSUES POTENTIALLY IMPACTING REUSE 
- N/A</gco:CharacterString>
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