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Sample GSM7839813

Query DataSets for GSM7839813

Status

Public on Feb 22, 2024

Title

DMN-EtFOSA-MP3CS46_L002

Sample type

SRA

Source name

RNA-Seq of daphnia magna whole body after 24 h exposure to N-EtFOSA-M

Organism

Daphnia magna

Characteristics

tissue: whole body
genotype: GLTED-wt
treatment: N-EtFOSA-M (DTXSID1032646)
conc.: 3
conc. units: ug/L
conc code: C
exposure duration: 24 h
age: 72-96h
replicate plate_id: P3

Treatment protocol

All assays were conducted in 96-well plate format using 1 ml volume deep-well plates (Catalog # 502162, NEST Scientific). Daphnia magna, one individual per well, were exposed to eight different concentrations of PFAS (two PFAS tested per plate) plus controls. Nominal PFAS concentrations ranged from 0.03 to 100 uM with 1/2 log spacing. Five replicate individuals were exposed to each treatment per plate, except control treatments (n=8). Uniform age (72 ± 6 h) Daphnia magna were manually loaded to each well of three replicate 96-well plates using a P1000 pipet with the end of the tip cut off to a one cm bore diameter. The volume in each well was reduced to a uniform volume of 50 µl using the Biomek i5 liquid handler. Assays were initiated by adding 550 µl of exposure solution from a dosing plate to the three replicate exposure plates for each assay. Immediately after dosing, each plate was covered with a silicone sealing mat (catalog #506065; NEST Scientific) and then placed in a 20°C incubator for 24 h. At test termination, each well was inspected and all mortalities (immobile with gentle prodding) were recorded and removed. Live daphnia were also examined for phenotypic abnormalities, although none were noted at sublethal concentrations. The Biomek i5 was then used to transfer 100 µl of exposure solution to an “exposure verification plate”, where the n=5 samples for each treatment concentration were subsequently pooled together into a total volume of 500 on a per-plate basis. The pooled exposure solution for each treatment was collected for quantitification of PFAS concentrations, with measured concentrations ultimately used for data analysis. The remaining volume of exposure medium in each well was replaced with 60 µl of Trizol (Zymo Research, CA, USA). A 2.3 mm stainless steel bead was added to each well, then the plate was homogenized at 30 Hz for two minutes using an Omni-Bead Ruptor bead mill. Homogenates of individual Daphnia magna from the same treatment group were pooled together, on a per plate basis to generate one pooled sample per treatment, per plate. Only live daphnia were included in the homogenate pools.

Growth protocol

Daphnia magna were obtained from an onsite culture unit at the U.S. EPA Great Lakes Toxicology and Ecology Division, Duluth, MN. For use in the assays, Daphnia magna were reared in mass culture in amended LSW and held at 20° C until they were 72 hours old. They were fed a diet of yeast-Cerophyll-trout chow and algae (USEPA 2016d) until ready for use in the assays. All procedures involving animals were approved by a local animal care and use committee.

Extracted molecule

total RNA

Extraction protocol

RNA was extracted from pooled homogenates using Zymo Research Direct-zol-96 RNA kit (Zymo Research) protocol adapted to the Biomek i5 to yield 40 µl of RNA. Previously extracted control RNA was added to empty wells on the final RNA plate. Concentrations of RNA for each well were measured on a NanoDrop spectrophotometer (Thermo Scientific, NanoDrop Eight) and diluted if necessary.
RNA extracts (10 µl) were used to prepare cDNA libraries with the Lexogen Poly(A) RNA Selection Kit V1.5 (catalog #157; Lexogen GmbH, Austria) and Lexogen CORALL mRNA-Seq Library Prep Kit with UDIs (catalog #158-163) both adapted to the Biomek i5. Resulting cDNA library samples were evaluated on a 4200 TapeStation (Agilent Technologies, CA, USA) using the D1000 ScreenTape (Agilent) analysis protocol to assess cDNA quality. Samples were quantified using the Quant-iT Picogreen dsDNA assay kit (catalog # P11495; Thermo Fischer Scientific), diluted if necessary, and equal portions of each sample pooled for use in RNA sequencing by Michigan State University.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

DM_N-EtFOSA-M_P3_C
DM_N-EtFOSA-M_counts.filtered.+1.normFinal.txt

Data processing

RNA sequencing was performed at the Michigan State University Research Technology Support Facility Genomics Core. Libraries were quality control checked and quantified using a combination of Qubit dsDNA HS, Agilent 4200 TapeStation HS DNA1000, and Invitrogen Collibri Library Quantification qPCR assays. Each pool (corresponding to ≈ 96 samples from assays with two PFAS) was loaded onto one lane of a NovaSeq S4 flow cell. Sequencing was performed in a 2x150bp paired end format using a NovaSeq 6000 v1.5 300 cycle reagent kit (target read depth of ≈28,000 MB/sample). Base calling was done by Illumina Real Time Analysis v3.4.4, and the output was demultiplexed and converted to FASTQ format with Illumina Bcl2fastq v2.20.0.
The Daphnia magna reference genome (assembly ASM2063170v1.1, RefSeq GCF_020631705.1) was indexed with STAR version 2.7.10a using the following sample specific parameters: --genomeSAindexNbases 12 --sjdbOverhang 149.
After performing quality control on the raw FASTQ data using FastQC and MultiQC version 1.12, raw reads were trimmed using Trim Galore! version 0.6.6, with the parameters --illumina --stringency 3 --quality 20 –e 0.1 --length 20 --paired, to remove sequencing adapters and low-quality bases and filter out trimmed reads shorter than 20 bp.
Trimmed reads were then mapped with STAR version 2.7.10a to the indexed genome using the default parameters except for the use of: --outSAMattributes NH HI AS NM XS. Samtools version 1.15.1 was used to sort and index the alignment files.
HTSeq version 2.0.1 was used to assign counts to each gene with the following parameters: -f bam -r pos -s no. FastQC and MultiQC version 1.12 were used to assess quality control metrics of the processed reads. Count data for treatment and plate-specific control samples for each PFAS were written to a single tab-delimited matrix file, where each column represented a sample and each row represented a gene.
Additional quality control and filter steps were applied to each count matrix using the R statistical computing software (R Core Team, 2022) prior to concentration response modeling. Lowly expressed genes were removed from the raw count matrices using edgeR’s (Robinson et al. 2010) built-in filtering function, filterByExpr(), in version 3.40.2; namely, genes with < 15 reads across all samples and < 10 reads in the smallest group (treatment or control) sample size were filtered out. Normalization factors for the filtered count matrices were calculated using the trimmed mean of M-values method via the edgeR function calcNormFactors() which was used for counts per million mapped reads (CPM) normalization. The filtered CPM normalized count matrix was log2 transformed with a pseudo-count of 1 added (to prevent occurrence of any negative count values for downstream analyses; i.e., log2(CPM+1)).
Assembly: Daphnia magna reference genome (assembly ASM2063170v1.1, RefSeq GCF_020631705.1)
Supplementary files format and content: Count matrices provided as tab delimited text files. Top row = sample names. Second row = treatment concentration (ug/L); all subsequent rows provide filtered counts per million (CPM) log2 transformed with a pseudo-count of 1 added (to prevent occurrence of any negative count values for downstream analyses; i.e., log2(CPM+1), with each row corresponding to a separate transcript and each column corresponding to a sample. One count matrix file per chemical exposure experiment.

Submission date

Oct 12, 2023

Last update date

Feb 22, 2024

Contact name

Daniel L. Villeneuve

E-mail(s)

villeneuve.dan@epa.gov

Organization name

US EPA

Department

Mid-Continent Ecology Division