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| Status |
Public on Mar 22, 2022 |
| Title |
zfish, embryo, 72hpf, 1b |
| Sample type |
SRA |
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| Source name |
whole embryo
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| Organism |
Danio rerio |
| Characteristics |
strain: AB
developmental stage: protruding mouth
treatment: 1 mg/L GenX
tissue: whole embryo
number of embryos: 9
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| Treatment protocol |
Undecafluoro-2-methyl-3-oxahexanoic acid (HFPO-DA, GenX (CASRN: 13252-13-6; Catalog No. 2121-3-13, SynQuest)) was purchased from SynQuest (Alachua, FL, USA, 97% purity). Exposure solutions were prepared one day before exposures by adding GenX to embryo water and stored at 4°C in a glass bottle. Since pH is known to affect embryo mortality 75, assays using pH neutralized GenX (titrated using NaOH and HCl, pH 6.6 – 7.6) were performed. Embryos were exposed to 0.5, 1, 2, 10, 1000, 4000, 6000, 8000, 10000, 12000, 16000, and 20000 mg/L titrated GenX concentrations from the sphere developmental stage (3 – 4 hpf) 49 until 72 hpf. Embryos were housed individually with 2 mL exposure solution in wells of 24-well plates. Negative controls were embryos grown in embryo water, while positive control embryos were exposed to 4 mg/L 3, 4-dichloroaniline. Fifty percent of the exposure solution in each well was refreshed daily. pH and dissolved oxygen (DO) for each plate were monitored and noted daily. Plates were discarded if more than 20% of embryos in plate controls died, or more than 20% of embryos in the negative control died. In the positive control, at least 30% mortality was necessary to maintain test validity 76.
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| Growth protocol |
Adult breeding populations of Danio rerio were maintained in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. AB strain adult zebrafish were obtained from Carolina Biological (Danio rerios, Burlington, NC, US) and acclimated to lab conditions for six months; embryos laid by the F1 generation were used for assays. Zebrafish were housed in a recirculating system in 1.3 or 3.3-liter polycarbonate tanks at a density of 10 fish/L and maintained at 28.5±1°C on a 14h light:10h dark cycle. Adults were fed twice daily with Tetramin® Tropical Flake Food (Tetra, Blacksburg, VA, US) in the morning (9 - 10AM) and freshly hatched brine shrimp (Brine Shrimp Direct, Ogden, UT, US) in the late afternoon (5 - 6PM) on weekdays and once daily with brine shrimp on weekends. Breeding tanks containing one female and two males were prepared one day before embryo collection. Following the onset of the light cycle, adult zebrafish were bred for one hour and embryos were collected in Pyrex glass dishes covered with mesh; embryos were staged per Kimmel et al. 49 under a stereo microscope (AmScope Compact Multi-Lens Stereo Microscope, model #SE306R-A) at 30X magnification. Debris and abnormally developing embryos exhibiting severe asymmetry in cleavage, detached cytoplasm, or broken chorions were removed from the batch before exposure. Developing embryos were kept in embryo water consisting of deionized water reconstituted with Instant Ocean® (Instant Ocean Spectrum Brands, Blacksburg, VA, US) to 60 μg/L, adjusted to pH 6.6 - 7.6 and 28.5°C until exposure.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was isolated from pooled samples of 8-10 embryos (four pools for each exposure) using the TRIzol reagent protocol with modifications (Invitrogen, Carlsbad, CA). Briefly, TRIzol was added to frozen embryos, before allowing embryos to thaw. Samples were homogenized in a Bead Mill 4 (Fisher Scientific, PA) using sterile 2 mm glass beads. RNA was resuspended in 50 µL RNase-free water. Quantity and quality of samples were determined using a NanoDrop 2000 (A260/280 and A260/230 > 1.8). Between 500 – 750 ng of total RNA per sample were sent to the Scripps Research Institute Next Generation Sequencing Core (TSRI, San Diego, CA) for shallow RNA-sequencing.
cDNA library preparation and shallow RNA-Seq was performed at TSRI. Sample quality was assessed using a 2100 Bioanalyzer 2100 (Agilent Technologies) (RIN>8.5). Library preparation for RNA-Seq was performed using the HTP RNA-Seq Library Prep Kit (iGenomX Inc, San Francisco, CA). Briefly, barcoded oligo dT primers were added to 50 ng RNA per sample, and reverse transcribed. Sample cDNA products were combined, cleaned, and run through PCR with 0.5 µM barcoded PCR primers (p5 and p7 sequences, Illumina, San Diego, CA). Purified PCR products were sequenced using a NextSeq2000 sequencer (Paired-end mode; read1: 26 bases, read2: 94bp) at TSRI. Greater than 1.5 million paired-end reads were generated for each sample.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 2000 |
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| Description |
Sample 10
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| Data processing |
Basecalls performed using Illumina NextSeq2000
The high throughput RNAseq data was initially processed with in-house custom scripts using BBTools to obtain demultiplexed reads for each sample based on the inline barcodes. The 5’-end 10-base barcodes and 3’-end 12-base UMI information from read1 fastq were first incorporated into read2 fastq as part of sequence ID using UMI-tools.
Raw RNA-Seq data quality was assessed using FastQCR (version 3.3), and UMIs were extracted using UMI-tools (version 1.1.2). To remove adapters and optimize for differential expression detection, Trimmomatic (version 0.40) was used to remove the first 16 bases of each read, trim reads according to a sliding-window of length 4 and minimum Phred score 20, and remove resulting reads shorter than 20 bases. SortmeRNA (version 2.1) was used to filter out contaminating rRNA.
The remaining reads were aligned to zebrafish genome assembly version 10 (GRCz10), due to lack of alignment-ready data for GRCz11. Reads were aligned using Spliced Transcripts Alignments to a Reference (STAR; version 2.7.3a). PCR deduplication was performed using extracted UMIs via UMI-tools (version 1.1.2)
Read counts per gene were quantified using HTSeq (version 0.13.5) with default settings
Transcript variants were compiled into single gene expressions. Gene expression for each library was normalized using trimmed mean of M values (TMM).
Count per million (CPM) filtering was performed from 0 to 5 CPM in increments of 0.25, and pairwise differential gene expression analysis was performed using the noiseqbio function of NOISeq (version 2.14.1) at each CPM threshold
Detected differentially expressed genes were plotted by CPM to select an optimal threshold for CPM filtering. Analysis proceeded with only genes of an average CPM above 2.75
Assembly: grcz10
Supplementary files format and content: fish_outlierrm_tmm.csv, lists for Samples 1 - 20 (column names), normalized and filter counts for gene (row names)
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| Submission date |
Mar 18, 2022 |
| Last update date |
Mar 22, 2022 |
| Contact name |
Goran Bozinovic |
| E-mail(s) |
goran@bozinstitute.org
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| Phone |
19196073155
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| Organization name |
Boz Life Science Research and Teaching Institute
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| Lab |
Boz
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| Street address |
3030 Bunker Hill St
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| City |
San Diego |
| State/province |
CA |
| ZIP/Postal code |
92109-5754 |
| Country |
USA |
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| Platform ID |
GPL30614 |
| Series (1) |
| GSE198976 |
Acute GenX exposure induces spinal deformations and arrhythmia, and alters expression of genes involved in cardiac, vascular, neural, and visual systems during zebrafish (Danio rerio) early development |
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| Relations |
| BioSample |
SAMN26800574 |
| SRA |
SRX14488037 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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