characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2018-05-22T17:03:04Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2017-08-25T10:56:47Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA104226513
characteristics.characteristic: External Id
characteristics.text: Stowers Institute for Medical Research
characteristics.characteristic: INSDC center name
characteristics.text: 2018-05-22T17:03:04Z
characteristics.characteristic: INSDC first public
characteristics.text: 2017-08-25T10:56:47Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS1885531
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-5952:O17-4.PE
characteristics.characteristic: Submitter Id
characteristics.text: 98 to 119
characteristics.unit: week
characteristics.characteristic: age
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: Ovaries were harvested from reproductively young and old mice in Leibovitz’s L-15 medium (Life Technologies Corporation, Grand Island, NY) supplemented with 3 mg/ml polyvinylpyrrolidone (Sigma-Aldrich, St. Louis, MO) and 0.5% Pen Strep (Life Technologies) (L15/PVP). Ovaries were disrupted mechanically using insulin syringes and late primary to transitioning secondary stage follicles were identified based on size and morphology and collected. For short-term culture (maximum of 40 minutes), follicles were cultured in CZB media (EMD Millipore, Billerica, MA) overlaid with mineral oil in a humidified atmosphere of 5% CO2 in air at 37 oC. Follicles were imaged using the EVOS FL Auto Cell Imaging system, and the mean diameter was determined based on two perpendicular measurements from basement membrane-to-basement membrane. Two age cohorts of adult CB6F1 female mice were used: 6-12 week old mice (reproductively young; Envigo, Indianapolis, IN) and 14-17 month old mice (reproductively old; National Institute on Aging Aged Rodent Colony, Bethesda, MD). 14-17 month old mice were selected as the reproductively old cohort because it has been shown that a significant deterioration in egg quality and the ovarian microenvironment occurs at this age (Chiang et al. 2010; Briley et al. 2016). We acknowledge that we do not know whether the founder sub-strains used to produce the F1 animals in our study were identical between our young and old mouse cohorts. Although we can not rule out confounding effects due to potential sub-strain variability, we think this contribution is likely to be minimal given that most reproductive aging phenotypes are conserved across strains (i.e. follicle loss, increased egg aneuploidy, increased ovarian stromal fibrosis) (Chiang et al. 2010; Lister et al. 2010; Hirshfeld-Cytron et al. 2011; Merriman et al. 2012; Briley et al. 2016). Pre-pubertal (12-13 day old) CD-1 female mice (Envigo) were used to optimize nucleolar antibody staining conditions. All mice were housed in a controlled barrier facility at the University of Kansas Medical Center’s (KUMC) Research Support Facility under constant temperature, humidity, and light (12h light/12h dark). Food and water were provided ad libitum. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Kansas Medical Center and were in accordance with National Institutes of Health Guidelines. Follicles were isolated from reproductive young (n = 5) and old CB6F1 (n = 4) mice as mentioned above. Isolated follicles were placed individually in 5 μl droplets of L15/PVP under oil and imaged immediately. Individual follicles were snap frozen in 3.5 μl 2x reaction buffer supplied in the cDNA synthesis reagents. For each animal, the total time that elapsed between mouse euthanasia and snap frozen samples did not exceed 30 minutes. Construction of cDNA was done using the Smart-Seq v4 Ultra Low Input RNA kit (Clontech Laboratories Inc, Mountain View, CA) with 14 cycles of PCR according to manufacturer’s instructions. Quality and yield of cDNA was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Adapter-ligated libraries were constructed from 1 ng cDNA using the Nextera XT kit (Illumina Inc, San Diego, CA) according to manufacturer’s instructions. Resulting libraries were checked for quality and quantity using the Agilent 2100 Bioanalyzer and Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA).
characteristics.characteristic: description
characteristics.text: normal
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000461
characteristics.characteristic: disease
characteristics.text: wild type genotype
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0005168
characteristics.characteristic: genotype
characteristics.text: O17
characteristics.characteristic: individual
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_10090
characteristics.characteristic: organism
characteristics.text: mature ovarian follicle
characteristics.characteristic: organism part
characteristics.text: reproductively old
characteristics.characteristic: phenotype
characteristics.text: follicle 4
characteristics.characteristic: sampling site
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: female
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000383
characteristics.characteristic: sex
characteristics.text: CB6F1
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0001339
characteristics.characteristic: strain
characteristics.text: O17-4.PE
characteristics.characteristic: title