Illumina HiSeq 2500 sequencing; Transcriptome analysis of self-renewing human pancreatic progenitors derived from pluripotent stem cells
Transcriptome analysis of self-renewing human pancreatic progenitors derived from pluripotent stem cells
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.
Institute of Medical Biology
E-MTAB-5731:Sample 4_s
Experimental Factor: cell line
AK6-13
Experimental Factor: cell type
cultured pancreatic progenitor cell (cPP)
Experimental Factor: passage
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