characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2018-01-14T17:01:58Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2017-09-12T18:52:29Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA104293468
characteristics.characteristic: External Id
characteristics.text: 1-Division of Pediatric General and Thoracic Surgery, Cincinnati Children’s Hospital Medical Center 2-
characteristics.characteristic: INSDC center name
characteristics.text: 2018-01-14T17:01:58Z
characteristics.characteristic: INSDC first public
characteristics.text: 2017-09-12T18:52:29Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS1918446
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-6017:tHIO+S_6441
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: H1
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0003042
characteristics.characteristic: cell line
characteristics.text: mid/hindgut intestinal organoid cell
characteristics.characteristic: cell type
characteristics.text: human
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: common name
characteristics.text: Protocols: Total tissue was collected in RLT Plus buffer (Quiagen) supplemented with 0.1% 2-Mercaptoethanol in FastPrep lysing Matrix D tubes. Human Intestinal Organoids (HIOs) were generated and maintained as previously described in Spence, J.R., Mayhew, C.N., Rankin, S.A., Kuhar, M.F., Vallance, J.E., Tolle, K., Hoskins, E.E., Kalinichenko, V.V., Wells, S.I., Zorn, A.M., et al. (2011). Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470, 105-109. Briefly, line H1 embryonic stem cells (WiCell Research Institute, Inc.) were grown in feeder-free conditions in Matrigel (BD Biosciences) coated six-well Nunclon surface plates (Nunc) and maintained in mTESR1 media (Stem Cell Technologies). For induction of definitive endoderm (DE), cells were passaged with Accutase (Stem Cell Technologies) and plated at a density of 65,000 cells per well in 24-well Nunc plates. Cells were allowed to grow in mTESR1 media for two days before treatment with 100 ng/ml of Activin A for three days as previously described. DE was then treated with hindgut induction medium (RPMI 1640, 100x NEAA, 2% dFCS,) for four days with 100 ng/ml FGF4 (R&D) and 3 µM Chiron 99021 (Tocris) to induce formation of mid-hindgut spheroids. Spheroids were then plated in Growth Factor Reduced (GFR) Matrigel and maintained in intestinal growth medium (Advanced DMEM/F-12, N2 supplement, B27 supplement, 15 mM HEPES, 2 mM L-glutamine, penicillin-streptomycin) supplemented with 100 ng/ml EGF (R&D) to generate human intestinal organoids (HIOs). Media was changed twice weekly thereafter. HIOs were replated in fresh Matrigel every 14 days. HIOs were prepared for transplantation as previously described in Watson, C.L., Mahe, M.M., Munera, J., Howell, J.C., Sundaram, N., Poling, H.M., Schweitzer, J.I., Vallance, J.E., Mayhew, C.N., Sun, Y., et al. (2014). An in vivo model of human small intestine using pluripotent stem cells. Nat Med 20, 1310-1314. Briefly, single matrigel embedded HIOs were transplanted into the mesentery of the mice. Mice were anesthetized with 2% inhaled isoflurane (Butler Schein), and the abdomen of the mouse was then shaved, and prepped in sterile fashion using isopropyl alcohol and povidine-iodine. A midline incision of approximately 2 cm was made in order for the intestine to be accessed. A small pocket was created in the mesentery and the HIO placed within. The skin was closed in a double layer and the mice were given a subcutaneous injection of Buprenex (0.05 mg/kg; Midwest Veterinary Supply) for pain management. Ten to twelve weeks following engraftment, the mice then underwent a secondary surgery with similar preparations. During this procedure, the engrafted tHIO was cut and briefly flushed before insertion of the gelatin capsule within the lumen of the tHIO. Mice were sacrificed and tissue harvested 14d postoperatively. Prior RNA extraction, total tissue collected in RLT Plus buffer were lysed using a FastPrep machine. Total RNA was extracted using an RNeasy Plus Micro Kit (Qiagen) according to manufacturer guidelines. The libraries were constructed according to manufacturer guidelines. (Illumina). Briefly, 350 to 900 ng of total RNA determined by Qbit high sensitivity spectrofluorometric measurement (Invitrogen) was poly-A selected and reverse transcribed using Illumina’s TruSeq RNA library preparation kit V2. Each sample was fitted with a unique adapter containing a 6 base molecular barcode for high level multiplexing. Prior sequencing, a 12 cycles of PCR amplification was performed on the samples.
characteristics.characteristic: description
characteristics.text: normal
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000461
characteristics.characteristic: disease
characteristics.text: organoid transplanted on NSG mouse mesentery
characteristics.characteristic: growth condition
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: organism
characteristics.text: mid/hindgut intestinal organoid
characteristics.characteristic: organism part
characteristics.text: embryonic stem cell
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CL_0002322
characteristics.characteristic: progenitor cell type
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: tHIO+S_6441
characteristics.characteristic: title