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Sample GSM7991432

Query DataSets for GSM7991432

Status

Public on Jul 20, 2024

Title

PFAS-8_replicate-3 [run66_s065]

Sample type

SRA

Source name

Zebrafish embyonic total RNA

Organism

Danio rerio

Characteristics

strain: Tropical 5D Wildtype
age: 48 hours post fertilization
pooled organisms: 10 whole embryos
treatment: 1H,1H,11H,11H-Perfluorotetraethylene glycol (PFAS-8)

Treatment protocol

At 6 hours post-fertilization (hpf), animals were statically exposed to chemicalsusing an HP D300 digital dispenser to dispense stocks into individual wells of the 96-well plates, all normalized to 1% DMSO, and plates were sealed.

Growth protocol

Animals were dechorionated at 4 hours post-fertilization (hpf) and then kept in 96-well plates in 100µLembryo medium consisting of 15 mM NaCl, 0.5 mM KCl, 1 mM MgSO4, 0.15 mM KH2PO4, 0.05 mM Na2HPO4, and 0.7 mM NaHCO3(Westerfield, 2000) at 28°C in the dark.

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated from pooled animals in Eppendorf safe-lock tubes by euthanizing on ice, immediately removing excess water, homogenizing in 200 µL RNAzol®RT (Molecular Research Center) with 100 µL 0.5 mm zirconium oxide beads (Next Advance) using a Bullet Blender® (Next Advance) at speed 8 for 3 minutes. After homogenization, 300 µL RNAzol® was added, and samples were stored at -80 °C. Isolation was performed using Direct-zol RNA MiniPrep kit (Zymo, Cat no. R2052) and optional DNase I digestion step.
RNA libraries for RNA-seq were prepared usingLexogen QuantSeq 3' mRNA-seq FWD kit following manufacturer's protocols.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

Total RNA isolated from zebrafish embryos treated with 53 µM 1H,1H,11H,11H-Perfluorotetraethylene glycol (PFAS-8; CAS 330562-44-2; 1% DMSO) from 6-48 hpf
2022MAR25All_Raw_Counts_Table_-_NOT_Concatenated_-_Sample_number_-_Experiments_A-B
FINAL_Raw_Counts_with_lowest_35th_percentile_genes_removed_-_Experiments_A-B
Differential_Gene_Expression_Experiments_A-B

Data processing

STAR for genome mapping and alignment (parameters: --outFilterType BySJout --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --outFilterMismatchNoverLmax 0.6 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --outSAMattributes NH HI NM MD --outSAMtype BAM SortedByCoordinate).
HTSeq for gene-level counts (parameters: -m intersection-nonempty -s yes -f bam -r pos).
For experiments A&B, genes with low counts were removed (i.e., genes with zero counts for all samples (9.9% of 32,521 genes), genes that were in the lowest 35th percentile of counts for all samples (4.8%)). Multidimensional scaling analysis in R (metaMDS function, vegan package) identified 1 outlier (PFAS-5_replicate-1) that was removed.
For experiment C, all genes with zero count were removed but low count genes were not removed. Control-C_48h_replicate-2 and Nafion byproduct 2_48h_replicate-3 were identified as outliers and removed.
DESeq2 for differential expression analysis (experiments A&B analyzed together, with sequencing runs collapsed by sample and samples contrasted to appropriate controls).
Assembly: GRCz11
Supplementary files format and content: .xlsx with read counts, filtered files and gene expression for all samples.

Submission date

Dec 26, 2023

Last update date

Jul 20, 2024

Contact name

Robyn Leigh Tanguay

E-mail(s)

[email protected]

Phone

5417376514

Organization name

Oregon State University