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Sample GSM5752795

Query DataSets for GSM5752795

Status

Public on Jan 10, 2022

Title

2128_Maternal_GD18 [SP0224-E12]

Sample type

SRA

Source name

Liver

Organism

Rattus norvegicus

Characteristics

tissue: Liver
treatment: Nafion byproduct 2
dose (mg/kg/d): 30
age: GD18
Stage: Maternal
strain: Sprague Dawley
study block: 143
dam id: 2128

Treatment protocol

Rat dams were exposed to NBP2 from GD14-18 (0, 0.1, 0.3, 1, 3, 10, or 30 mg/kg/d, n = 4 per dose except n = 6 for controls) or GD17-21 (0, 1, 3, 10, or 30 mg/kg/d, n = 3 per dose). Dams and fetuses were euthanized by decapitation 2–4 h after the final oral dose (∼08:30–10:30 EST). Euthanasia order was stratified such that the timing of necropsy was equally distributed across dose groups. Maternal and fetal liver mRNA underwent targeted RNA-sequencing.

Growth protocol

Time-mated Sprague-Dawley rats (Crl:CD(SD)), approximately 90 days old, were purchased from Charles River Laboratories (Raleigh, NC, USA) and shipped to USEPA (Research Triangle Park, NC, USA) on GD2 (GD0 = bred date; GD1 = plug positive date). Dams and their offspring were housed individually in clear polycarbonate cages (20 × 25 × 47 cm) with heat-treated, laboratory-grade pine shavings (Northeast Products, Warrensburg, NY) and fed NIH07 Rat Chow and filtered (5 µm) municipal tap water (Durham, NC) ad libitum. Dams were weight-ranked and stratified then randomly assigned to treatment groups to produce similar mean weights and variances given the range of dam body weights (typically ∼10% coefficient of variation). This study was conducted in accordance with a protocol approved by the USEPA Center for Public Health and Environmental Assessment Institutional Animal Care and Use Committee. Animals were housed in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care and maintained at 20–22 °C, 45–55% humidity, and a 12:12 h photoperiod (06:00 – 18:00 EST).

Extracted molecule

total RNA

Extraction protocol

Subsamples of liver tissue (∼30–50 mg) were collected from NBP2-exposed dams (GD18 and GD21) and fetuses (GD18 and GD21) were placed in polypropylene microcentrifuge tubes containing 500 µL TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) on ice, then individually homogenized using a Bullet Blender (Next Advance, Troy, NY, USA) with 1 mm zirconium oxide beads and stored at −80 °C prior to RNA extraction. RNA extraction was conducted according to TRIzol Reagent manufacturer specifications using chloroform and isopropanol. Following extraction, RNA was purified using RNeasy Mini Kit (Cat no. 74104; Qiagen, Hilden, Germany). RNA concentration and purity (260:280 ratio ≥ 1.8) were determined with a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA).
Libraries were prepared as described in Trejo et al. (2019) (https://doi.org/10.1371/journal.pone.0212031). Purified RNA was processed in annealing buffer with detector oligos (DOs), probes that have high selectivity for 21,119 protein coding genes in the rat genome (Rat Whole Transcriptome Assay on the Templated Oligo Detection Assay platform; TempO-Seq, BioSpyder Technologies, Carlsbad, CA, USA). After oligo ligation, a nuclease mixture was added to digest unbound or erroneously bound DOs. A final ligase mix was added to facilitate addition of a single primer pair. The ligases were deactivated with heat and the probes were amplified by PCR, which added sample specific barcodes that flank the target sequence and are inserted into the standard Illumina adaptors. PCR-amplified and barcoded samples were then pooled into a single library, purified and sequenced. TempO-Seq RNA samples were sequenced on the Illumina HiSeq 2500 platform with 50-bp single-end reads to a target read depth of 5 million. Overly abundant transcripts were attenuated equally across all samples to save sequencing reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Sequencing alignment and data analysis were conducted using Partek Flow build 9.0.20.1104 (Partek Incorporated, St. Louis, MO, USA)
Raw FASTQ files provided from BioSpyder were uploaded into Partek Flow and aligned to the reference genome rn6 using STAR v.2.7.3a with default settings.
Aligned reads were then quantified to rn6-Ensembl Transcripts release 100 by Partek Expectation Maximization. Following quantification, low expression gene features with a mean count ≤ 10 across all samples were removed, a 0.0001 prior count was added to adjust for zero counts in the dataset, samples were normalized using count per million mapped reads (CPM), and finally log2 transformed.
Samples were separated by life stage into four groups: maternal GD18, maternal GD21, fetal GD18 and fetal GD21. Principle component analysis of sample gene counts from each group revealed three outlier samples (F07, B02, and G07) which were removed prior to downstream gene expression analysis.
Differentially expressed genes (DEGs) were identified for each life stage using Partek Gene Specific Analysis algorithm (GSA) on pairwise groups (Treated vs. vehicle control). GSA fit data from each gene feature to multiple models: negative binomial, log-normal, normal, and log-normal with shrinkage models. The best fit model was selected for each feature based on the lowest Akaike Information Criterion value. Significant DEGs for each life stage were identified based on a false discovery rate (FDR) adjusted p-value < 0.05 (Benjamini and Hochberg 1995) and an absolute fold change ≥ 2.
Genome_build: rn6, Ensembl build 100
Supplementary_files_format_and_content: raw count matrix in .txt format
Supplementary_files_format_and_content: Low expression filtered, 0.0001 count offset added, counts per million (CPM) normalized, and log2 transformed count matrix prior to outlier removal in .txt format

Submission date

Dec 24, 2021

Last update date

Jan 10, 2022

Contact name

Leah Wehmas

Organization name

US EPA

Street address

109 T.W. Alexander Dr.

City

Durham