characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-04-19T00:19:05Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-04-19T00:19:05Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA112482435
characteristics.characteristic: External Id
characteristics.text: 1) Department of Morphology, Biomedical Research Institute, Faculty of Medicine and Life Sciences, Hasselt University, Belgium 2) Laboratory of Tissue Plasticity in Health and Disease, Cluster of Stem Cell and Developmental Biology, Department of Development and Regeneration, KU Leuven (University of Leuven), Leuven, Belgium
characteristics.characteristic: INSDC center name
characteristics.text: 2023-04-19T00:19:05Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-04-19T00:19:05Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS14599786
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12557:FH118
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: MTO
characteristics.characteristic: cell_type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: Early-passage (P0) molar- and incisor tooth organoids (MTO, ITO respectively) grown in TOM with or without exogenous EGF, or P5 MTO and ITO grown in TOM+EGF and exposed to DM or not (n=3 biological replicates per condition) were harvested from Matrigel droplets using ice-cold serum-free defined medium (SFDM; DMEM/F12 1:1, 5 mg/l human transferrin, 5mg/l insulin Hybrimax from bovine pancreas, 35mg/L penicillin, 50mg/L streptomycin, 600uL/L ethanol, 50µl/L catalase, 1g/L NaHCO3, 0.5% BSA). Collected samples were lysed with lysis buffer from the GenElute Mammalian Total RNA Miniprep kit (Sigma-Aldrich) following manufacturer’s instructions. C57BL/6 postnatal day 7 pups were euthanized by decapitation, after which the mandibles were collected in Dulbecco’s Modified Eagle Medium with 10 mM HEPES (DMEM; Gibco), supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich) and 1% penicillin-streptomycin (Gibco). Whole (unerupted) molars, including surrounding dental follicle and attached epithelium, as well as apical ends of (unerupted) incisors were carefully isolated, rinsed with phosphate-buffered saline (PBS; Gibco) and incubated with 2.5% Trypsin (Gibco) for 30 minutes at 37°C. Following trypsinization, the dental follicle and attached epithelium were manually recovered from the molars, and the dental pulp was separately collected from the molar pulp chamber. Subsequently, the molar (dental follicle and attached epithelium) and incisor tissues were further enzymatically digested with TrypLE Express (Gibco) supplemented with 5 μM ROCK inhibitor (ROCKi, Y-27632; Thermo Fisher) for 15 minutes at 37°C, and mechanically triturated (using syringes with decreasing diameters: 18G (Terumo), 20G (BD) and 26G (BD)) to a suspension containing single cells and small cell clusters. The number of collected cells was determined with a Z2 Coulter Particle Count and Size analyzer using COULTER Z2 AccuComp software (Beckman Coulter). The dissociated molar and incisor dental epithelial cell material was resuspended in SFDM and growth factor-reduced Matrigel (Corning) at a 30:70 ratio, supplemented with 10 μM ROCKi. Cells were seeded at 20,000 and 12,500 cells per 20 μL drop from molars and incisors, respectively. After solidification, pre-warmed TOM (SFDM supplemented with with 0.5 uM A83-01, 1X B27, 100 ng/mL cholera toxin, 20 ng/mL bFGF, 200 ng/mL FGF8, 100 ng/mL FGF10, 2 mM L-glutamine, 100 ng/mL IGF1, 1X N2, 1.25 mM N-acetyl L-cysteine, 10 mM Nicotinamide, 100 ng/mL noggin, 200 ng/mL RSPO1, 10 uM SB202190, 100 ng/mL SHH and 200 ng/mL WNT3) was added. Organoid cultures were kept at 37 °C in a 1.9% CO2 incubator (as required by the SFDM buffering system), and medium was refreshed every 2 days. To passage the TO as occurring every 7-10 days, Matrigel droplets were collected using ice-cold SFDM, followed by incubation with TrypLE containing 5 μM ROCKi and mechanical trituration to dissociate the organoids. Remaining large organoid fragments were allowed to sediment, and the cell number in the supernatant, containing single cells and small fragments, was determined as described above. Upon reaching stable growth, dissociated MTO and ITO were seeded at 15,000-20,000 cells and 7,500 cells per 20 μL droplet, respectively. Early passage organoids (passage 0) for characterization were grown in TOM or TOM supplemented with 20 ng/mL EGF. Late passage organoids (passage 5) for differentiation experiments were grown in TOM supplemented with 20 ng/mL EGF for 7 days before medium was switched to differentiation DM (SFDM supplemented with 1X B27, 100 ng/mL BMP2, 50 ng/mL BMP4, 20 ng/mL EGF, 20 ng/mL bFGF and 4 ng/mL TGF-beta1) for 7 more days before collection. Total RNA was isolated using the GenElute Mammalian Total RNA Miniprep kit (Sigma-Aldrich) following manufacturer’s instructions, and RNA Integrity Number (RIN) determined with Agilent Picochips on an Agilent BioAnalyzer 2100 (Agilent Technologies). Samples with RIN > 7.5 were sequenced. TruSeq total stranded RNA library preparation was performed.
characteristics.characteristic: description
characteristics.text: PD7
characteristics.characteristic: developmental stage
characteristics.text: normal
characteristics.characteristic: disease
characteristics.text: wild type genotype
characteristics.characteristic: genotype
characteristics.text: DM
characteristics.characteristic: growth condition
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: molar
characteristics.characteristic: organism part
characteristics.text: P5
characteristics.characteristic: passage
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: FH118
characteristics.characteristic: title