characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2017-06-06T17:01:36Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2017-05-16T18:32:04Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA104054743
characteristics.characteristic: External Id
characteristics.text: unspecified
characteristics.characteristic: INSDC center name
characteristics.text: 2017-06-06T17:01:36Z
characteristics.characteristic: INSDC first public
characteristics.text: 2017-05-16T18:32:04Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS1718636
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-5731:Sample 1
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: H9
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0003045
characteristics.characteristic: cell line
characteristics.text: cultured pancreatic progenitor cell (cPP)
characteristics.characteristic: cell type
characteristics.text: human
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: common name
characteristics.text: Protocols: Pluripotent cell lines were obtained as follows: H9 hESC were purchased from WiCell (WA09), HES3 hESC were derived in house from human embryos, and the hiPSC line AK6-13 was derived in house from human fibroblasts. Human pluripotent stem cells were maintained in mTeSR1 media on Matrigel-coated tissue culture plastic, and differentiated into pancreatic progenitors using the STEMdiff Pancreatic Progenitor kit (STEMCELL Technologies, 05120) according to the manufacturers instructions with the following modifications: (1) cells were initially seeded into 12 well plates (Corning, 353043) at a density of 106 cells/well, and (2) stage 1 was extended to last 3 days - rather than 2 - by repeating the final day’s treatment. To generate cultured Pancreatic Progenitor (cPP) cell lines, day 15 differentiation cultures (PPd15 cells) were treated with Accutase to generate single cells, which were then replated onto a layer of lethally irradiated 3T3-J2 feeders (0.5-1x106 cells/cm2) in media composed of the following: advanced DMEM/F12 (Thermo Fisher Scientific, 21634010), 2mM L-glutamine (Thermo Fisher Scientific, 25030), 100U/mL penicillin/streptomycin (Thermo Fisher Scientific, 15140122), 1X N2 supplement (Thermo Fisher Scientific, 17502-048), 1X B27 supplement (Thermo Fisher Scientific, 17504-044), ), 30nM dexamethasone (STEMCELL Technologies, 72092), 50ng/mL EGF (R&D Systems, 236-EG-200), 50ng/mL FGF10 (Source Bioscience, ABC144), 3μM retinoic acid (RA) (Sigma, R2625), 10μM SB431542 (Calbiochem, 616464), and 1μM DAPT (Sigma, D5942). Gentle cell dissociation reagent (STEMCELL Technologies, 07174) was used to passage cPP cells as aggregates which were then seeded at a 1:6 split ratio. All tissue culture was carried out in a 5% CO2 air environment at 37oC. RNA was isolated from samples harvested from cPP and PPd15 cultures using an RNeasy mini kit (Qiagen, cat # 74104). Feeder removal microbeads (Mintenyi, 130-095-531) were used to deplete cPP cells of 3T3 feeders prior to RNA extraction. All RNA samples had an RNA integrity number > 9. RNA-seq libraries were generated using the NEBNext® Ultra™ RNA Library Prep Kit, (NEB, E7530L). Messenger RNA was enriched by hybridization to oligo-dT.
characteristics.characteristic: description
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: 8
characteristics.characteristic: passage
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: Sample 1
characteristics.characteristic: title