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Sample GSM4970429

Query DataSets for GSM4970429

Status

Public on Oct 04, 2022

Title

A15247_Hippocampus(N975)

Sample type

SRA

Source name

Hippocampus

Organism

Macaca fascicularis

Characteristics

individual: A15247
weight: 3.58 kg
age: 6.35 years
Sex: Female
tissue: Hippocampus
dose: 0 (mg/kg/day)
total dosing time: 653 days

Treatment protocol

Animals were fasted for 12 hrs and sedated under 20 mg/kg ketamine, before transportation to the necropsy room at the WaNRPC. Sedated animals were euthanized with an intravenous overdose of sodium pentobarbital, as per the AMVA Panel on Euthanasia recommendations (Leary et al., 2020). Euthanasia was confirmed and the brain quickly excised from the skull. The brain was bisected along the midsagittal plane. ROIs, including the HIP and THA, were identified by a certified veterinary pathologist. From the left hemisphere, dissected tissue was sliced into small, approximately 3 cm sections (representing 1-3 sections/ROI), placed in a 2 ml cryostat tube, immediately frozen in liquid nitrogen, and stored at -80°C.

Growth protocol

Adult female fascicularis macaque monkeys between 5.5-11 years of age (mean: 7 years) and 2.8-4.2 kg in body weight (mean: 3.5 kg) were enrolled in the original study designed to assess the reproductive and neurodevelopmental effects of exposure to low-level DA (Burbacher et al., 2019). Animals were singly housed in stainless steel caging equipped with metabolic pans and a wide mesh that provided daily contact with an adjacent social partner in the Infant Primate Research Laboratory at the Washington National Primate Research Center (WaNPRC) in Seattle, WA, USA. The room was maintained 24 ± 4°C on a 12-hr light/dark schedule. Animals were given Lab Diet High Protein Monkey Diet (St. Louis, MO, USA) biscuits 2x/day, regular environmental enrichment (fresh and frozen fruit, vegetables, cereals, toys, puzzle balls, etc.) daily, and provided filtered water ad libitum. All research protocols strictly adhered to the guidance of the Animal Welfare Act and the Guide for Care and Use of Laboratory Animals of the National Research Council and were approved by the University of Washington Institutional Animal Care and Use Committee. Observers were experimentally blinded to the exposure conditions of the macaques and employed positive reinforcement techniques to train animals to drink out of a syringe to allow for controlled dosing of DA or vehicle control. Following training, animals underwent a baseline period of assessment designed to asses clinical signs of toxicity prior to starting daily oral exposure of 0 (n=10), 0.075 (n=11), or 0.15 (n=11) mg/kg of DA (BioVectra, Charlottetown, PE, Canada) in 5% sucrose water. Animals continued to be assessed 3-5x per week throughout a 2-month pre-breeding period, a 1-8-month breeding period, a 6-month pregnancy, and a 2-12-month postpartum period. Exposure ceased postpartum for 8 animals, and for 24 animals dosing continued until necropsy. In all animals, behavioral assessment continued at least 3x/week until necropsy.

Extracted molecule

total RNA

Extraction protocol

Between 30-50 mg of amygdala, hippocampus, or thalamus of non-human primate tissues were added to 1mL TRIzol with 5mm stainless steel beads (Qiagen, Germantown, MD). Tissues were homogenized using Qiagen’s TissueLyser LT for 2 mins at 40Hz. Homogenized tissues were centrifuged at 10,000 RPM for 2 mins. The supernatant was transferred to a new tube and an equal volume of 100% ethanol was added. Total RNAs, including small and micro RNAs, were extracted using Direct-zol RNA Miniprep Plus (Zymo Research, Irvine, CA) according to manufacturer’s protocol. Extracted RNAs were quantified on a Qubit 4 Fluorometer using the RNA HS Assay Kit (Thermo Fisher Scientific, Grand Island, NY) and qualified using a Bioanalyzer 2100 (Agilent, Santa Clara, CA).
Total RNA is normalized to 7.5ng/ul in a total volume of 50ul on the Perkin Elmer Janus Workstation (Perkin Elmer, Janus II). Poly-A selection and cDNA synthesis are performed using the TruSeq Stranded mRNA kit as outlined by the manufacturer (Illumina, cat#RS-122-2103). All steps are automated on the Perkin Elmer Sciclone NGSx Workstation to reduce batch to batch variability and to increase sample throughput. Final RNASeq libraries are quantified using the Quant-it dsDNA High Sensitivity assay, and library insert size distribution is checked using a fragment analyzer (Advanced Analytical; kit ID DNF474). Samples where adapter dimers constitute more than 4% of the electropherogram area are failed prior to sequencing. Technical controls (K562,Thermo Fisher Scientific, cat# AM7832), are compared to expected results to ensure that batch to batch variability is minimized. Successful libraries are normalized to 10nM for submission to sequencing.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

N975
counts.txt

Data processing

FASTQ files were aligned against the NCBI M. fascicularis transcriptome using the salmon aligner (v1.2.1), using the following arguments: --validateMappings --incompatPrior 1 -p 30 --useVBOpt --gcBias --posBias --biasSpeedSamp 10
Transcript counts were read into R using the Bioconductor tximport package, summarizing transcript (NCBI RefSeq transcript) counts at the gene (NCBI Gene ID) level using length scaled TPM abundances (e.g., using countsFromAbundance = “lengthScaledTPM”).
Genome_build: GCF_000364345.1 (Macaca_fascicularis_5.0, downloaded on 22 November 2019).
Supplementary_files_format_and_content: counts.txt: Matrix of read counts per gene. The first column is the NCBI Gene ID, and the remaining columns are the counts/gene for each sample. First row lists the sample IDs.

Submission date

Dec 10, 2020

Last update date

Oct 04, 2022

Contact name

James William MacDonald

E-mail(s)

jmacdon@uw.edu

Organization name

University of Washington

Department

Environmental and Occupational Health Sciences