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Sample GSM5632838

Query DataSets for GSM5632838

Status

Public on Oct 10, 2022

Title

8_1_control_FFPE

Sample type

SRA

Source name

formalin-fixed paraffin embedded left lateral and right media liver lobes

Organism

Mus musculus

Characteristics

tissue: liver
strain: B6C3F1
age: 4 weeks
Sex: male
treatment: dichloroacetic acid (DCA)
dose: 0 mg/kg-d

Treatment protocol

Dichloroacetic acid (CAS 79-43-6, Aldrich) was administered in the drinking water at doses of 0, 1.0, 2.0, and 3.5 g/L (as described in DeAngelo et al. 1991) for six days. Drinkin water consumption along with measured DCA dose-levels were converted to mg per kg bodyweight per day (mg/kg-d) as 0, 198, 313, and 427 mg DCA/kg-d

Growth protocol

Studies were conducted in 4 week-old male B6C3F1 mice maintained under standard housing conditions at the U.S. Environmental Protection Agency (US EPA) AAALAC-accredited animal facility (Research Triangle Park, NC). Animal care and procedures were conducted under protocols approved by the Institutional Animal Care and Use Committee of the USEPA.

Extracted molecule

total RNA

Extraction protocol

RNA from FROZ samples was isolated using homogenization and RNAzol® RT (cat #RN 190; Molecular Research Center, Cincinnati, OH) followed by purification using a RNeasy MinElute column (Qiagen, GmbH, Hilden, Germany). RNA purity and concentration were assessed by Agilent Bioanalyzer (RNA 6000 Pico Kit cat #5067-1514 or Nano Kit 5067-1529; Agilent Technologies GmbH, Berlin, Germany), NanoDrop™-2000 spectrophotometer (ThermoFisher Scientific, Waltham, MA) and Qubit fluorometer (RNA BR Assay Kit cat #Q10210 or HS Assay Kit cat #Q32852; ThermoFisher Scientific, Waltham, MA). For FFPE samples, excess paraffin was removed and the tissue was transferred into lysis buffer, melted in mineral oil to remove remaining paraffin, and lysed. RNA within the lysate is annealed to a matched pair of detector oligos, with each pair of detector oligos targeting a single transcript within the mouse genome.
Libraries were prepared as described in Trejo et al. (2019) (https://doi.org/10.1371/journal.pone.0212031). FROZ RNA or FFPE lysate was processed in annealing buffer with detector oligos (DOs), probes that have high selectivity for 21,451 Mus musculus target genes (Whole Genome Mouse Assay, BioSpyder Technologies; Carlsbad, CA). 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.
Targeted RNA-seq

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

8_FFPE

Data processing

FASTQ files uploaded to PartekFlow (Partek build version 9.0.20.0202, St. OLouis, MO)
Reads mapped to mm10 using Star 2.6
Quantitation (Partek expectation maximization) to mm10 - RefSeq Transcripts 92
Raw gene counts were offset by a 0.0001 count, filtered to remove any gene feature with geometric mean across all samples <1, counts per million mapped reads (CPM) normalized and log2 transformed
Significant differentially expressed genes (DEGs) were identified using Partek Gene Specific Analysis (GSA) fit to multiple models (negative binomial, normal and log-normal). The lowest corrected Akaike Information Criterion (AIC) was used to select the gene model fit. Significant DEGs were defined as genes with false discovery rate (FDR) adjusted p-value <0.05 (Benjamini and Hochberg 1995) and absolute fold change > 2.
Genome_build: MM10
Supplementary_files_format_and_content: tab deliminated files with filtered, counts per million mapped reads normalized, and log2 transformed counts for all genes

Submission date

Oct 19, 2021

Last update date

Oct 10, 2022

Contact name

Leah Wehmas

Organization name

US EPA

Street address

109 T.W. Alexander Dr.

City

Durham