Satomi Kohno Thea M Edwards 2025 tabular digital data Reston, VA U.S. Geological Survey Authors Open Researcher and Contributor Id (ORCID) are as follows: Satomi Kohno: 0000-0001-6184-3616; Thea M Edwards: 0000-0002-6176-2872 https://doi.org/10.5066/P13YCQOV These RNA sequencing (RNA-seq) data were collected to better understand how exposure to environmentally relevant doses of nitrate affects the brain transcriptome in fathead minnows. Fish were exposed to nominal doses of 0, 2, 5, 10, 25, or 100 mg/L nitrate nitrogen (NO3-N), added to flow-through well water as sodium nitrate. Transcriptomic data were collected from three experiments that addressed different life stages and durations of nitrate exposure: 1) acute exposure of juvenile fathead minnows for four days, from 75 to 79 days post fertilization (dpf), immediately before sexual maturation was expected to occur; 2) chronic exposure of juvenile fathead minnows from fertilization (day 0) through 79 dpf; this window covers all developmental stages until the beginning of sexual maturation; and 3) acute exposure of reproductive male and female pairs through one spawning cycle. The adult exposure occurred after fish were observed for two spawning cycles in clean water to obtain baseline data on their reproductive cyclicity, fecundity, and fertility. After the exposures, fish were euthanized and dissected to collect whole brains. Brain RNA was extracted, sequenced, and analyzed to identify genes and gene pathways that were affected by nitrate to improve our understanding of how nitrate affects fish physiology. Additionally, RNA data were analyzed to identify possible genetic biomarkers of nitrate exposure. The life history and water quality data for these studies is released in a separate but linked data release. Data were collected to assess 1) the effects of long and short-term nitrate exposure on the brain transcriptome of juvenile fathead minnows, and 2) the effects of short-term nitrate exposure, through one spawning cycle, on the brain transcriptome of adult male and female fathead minnows. Nitrate exposure levels represent the range of nitrate that occurs in rivers and lakes that receive nitrate inputs from agricultural or municipal sources. Transcriptomic data are needed to better understand how nitrate exposure influences gene expression in the brain. Changes in the brain transcriptome could explain other changes in downstream physiology and endocrinology that have been previously published for fishes exposed to nitrate. The data advance our understanding of nitrate ecophysiology. 20190916 20200305 ground condition Complete None planned Columbia Environmental Research Center -92.284312 -92.274450 38.912612 38.910784 ISO 19115 Topic Category farming USGS Thesaurus nitrogen fish laboratory experiments Biocomplexity Thesaurus transcripts nitrates USGS Metadata Identifier USGS:1dbd75f0-6aee-4859-b252-17a9e944a054 Geographic Names Information System Missouri None Columbia Environmental Research Center Integrated Taxonomic Information System Pimephales promelas Kingdom Animalia animals Subkingdom Bilateria triploblasts Infrakingdom Deuterostomia Phylum Chordata chordates Subphylum Vertebrata vertebrates Infraphylum Gnathostomata Superclass Actinopterygii ray-finned fishes spiny rayed fishes Class Teleostei Superorder Ostariophysi Order Cypriniformes minnows suckers Superfamily Cyprinoidea Family Leuciscidae Subfamily Pogonichthyinae Genus Pimephales bluntnose minnows Species Pimephales promelas Fathead Minnow TSN: 163517 None. Please see 'Distribution Info' for details. It is requested that the authors and the USGS Columbia Environmental Research Center be cited for any subsequent publications that reference this dataset. Users are advised to read the dataset's metadata thoroughly to understand appropriate use and data limitations. Thea M Edwards U.S. Geological Survey, Columbia Environmental Research Center Supervisory Research Biologist mailing and physical

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Columbia MO 65201 United States 573-875-5399 tedwards@usgs.gov Rachel Claunch, James Candrl, John Carroll, Vanessa Velez, Daniel Lamm, and Lillie Bradshaw assisted with sampling during the experiment. Rachel Claunch and Lillie Bradshaw assisted with data management and curation. Windows 11 version 23H2, build 22631.3880; notepad++ 64-bit x64 version 8.6.9 Two personnel double-checked the sample names and sequence file names. Since the RNA-Seq data are not human-readable, the quality was checked using the FastQC program (https://github.com/s-andrews/FastQC). Two personnel double-checked the sample names and sequence file names. Adapter and low-quality sequences were filtered out using Cutadapt, https://github.com/marcelm/cutadapt/, a paired read sequence with a quality cutoff of 20 and minimum length of 20. Data set is considered complete for the information presented, as described in the abstract. Users are advised to read the rest of the metadata record carefully for additional details. No formal positional accuracy tests were conducted No formal positional accuracy tests were conducted Bérénice Batut Mallory Freeberg Mo Heydarian Anika Erxlebe Pavankumar Videm Clemens Blank Maria Doyle Nicola Soranzo Peter van Heusden Lucille Delisle 2020 application/service https://gxy.io/GTN:T00295 Digital and/or Hardcopy 2020 publication date Batut et al. Methodology used for processing RNA-seq data Marcel Martin 20110502 publication EMBnet.journal vol. 17, issue 1 n/a EMBnet Stichting ppg. 10 https://doi.org/10.14806/ej.17.1.200 Digital and/or Hardcopy 2011 publication date Cutadapt Tool used for filtering out adapter and low-quality sequences Simon Andrews 20100426 application/service https://github.com/s-andrews/FastQC https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ Digital and/or Hardcopy 2010 publication date FastQC Program used to perform quality control on sequence data This is RNA sequencing (RNA-seq) data to assess the effects of nitrate exposure on the brains of reproductive adult and juvenile fathead minnows. All exposures were conducted in glass fish tanks served with flow-through well water that was warmed to 24 degrees Celsius; the light cycle was 16 hours of light and 8 hours of dark. Fish were fed ad libitum with brine shrimp for the first 10 days post hatching, then brine shrimp plus flake food for the next 20 days, then flake food and ground trout chow after 20 days. For all nitrate exposures, sodium nitrate was continuously dosed into flow-through well water to achieve nominal doses of 0, 2, 5, 10, 25, or 100 mg/L nitrate nitrogen (NO3-N). For the adult exposures, 16 adult male and female pairs were housed individually with a spawning hut in a 5-gallon glass tank. Pairs were acclimated in their tanks for two spawning cycles. On the day after their second spawning, sodium nitrate was added into the flow-through well water; exposure continued through the third spawning cycle plus one day. On the day after the third spawning, the adult pair was dissected, and whole brains were refrigerated in RNAlater for 24 hours and then stored at -80 degrees Celsius. For the juvenile exposures, two experiments (acute and chronic) were conducted concurrently on the same cohort of fish. Fertilized eggs were obtained from 12 breeding pairs and mixed together. For the acute juvenile exposure, approximately half of the fertilized eggs were raised in clean flow-through well water for 75 days post fertilization (dpf) and then exposed to nitrate for 4 days from 75 through 79 dpf. For the chronic juvenile exposure, approximately half of the fertilized eggs were raised with nitrate from 1 dpf through 79 dpf. At 79 dpf, juvenile fish from both acute and chronic studies were dissected to collect whole brains, which were refrigerated in RNAlater for 24 hours and then stored at -80 degrees Celsius. 20200104 Total RNA was extracted using the RNeasy kit (Qiagen), and the RNA quality and quantity were evaluated using the QIAxcel RNA kit. 20200903 The RNA-Seq library was prepared from mRNA using a TruSeq Stranded mRNA kit (Illumina), and the library was analyzed in paired-read sequencing for 100-bp with 50 million reads per sample in the NovaSeq 6000 S4 Flow Cell system (Illumina). 20210617 Adapter and low-quality sequences were filtered out using Cutadapt, https://github.com/marcelm/cutadapt/, a paired read sequence with a quality cutoff of 20 and minimum length of 20. 20211007 This data release is made up of a number of files in Fastqsanger.gz format. These are stored on the NCBI Sequence Read Archive (SRA, https://www.ncbi.nlm.nih.gov/sra), under bioproject PRJNA1220128: Effects of aquatic nitrate on brain transcriptome in fathead minnows. Biosample accession numbers are SAMN46710669 through SAMN46710764. Kohno, S, and Edwards, T.M. 2025. Effects of aquatic nitrate exposures in the Laboratory on brain transcriptome in fathead minnows: U.S. Geological Survey data release, https://doi.org/10.5066/P13YCQOV GS ScienceBase U.S. Geological Survey mailing and physical

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Denver CO 80225 United States 1-888-275-8747 sciencebase@usgs.gov Unless otherwise stated, all data, metadata and related materials are considered to satisfy the quality standards relative to the purpose for which the data were collected. Although these data and associated metadata have been reviewed for accuracy and completeness and approved for release by the U.S. Geological Survey (USGS), no warranty expressed or implied is made regarding the display or utility of the data on any other system or for general or scientific purposes, nor shall the act of distribution constitute any such warranty. 20250303 CERC Data Manager U.S. Geological Survey, Columbia Environmental Research Center Natural Resource Data Manager mailing and physical

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Columbia MO 65201 United States 573-875-5399 gs-mw-cerc_data_manager@usgs.gov FGDC Biological Data Profile of the Content Standard for Digital Geospatial Metadata FGDC-STD-001.1-1999