strain: 129X1/SvJ x 129S1/SV-+p+Tyr- cKitlSl-J/+ tissue: in vitro derived definitive endoderm sentrix_id: 8615501026 sentrix_position: C
Mouse PSC Differentiation: Maintenance cultures were split and MEF depleted for 30 minutes in maintenance media. PSCs were collected and resuspended in differentiation media 1 [A-DMEM (GIBCO, 12491) supplemented with 1% N2 (GIBCO, 17502-048), 2% B27 minus vitamin A (GIBCO, 12587-010), 1% Glutamax (GIBCO, 35050079) 1% P/S (Cellgro, 30-001-CI)] and 10µM Y-27632 (Selleck Chemicals, S1049). Cells were plated at 5x103 cells/cm2 onto 0.1% gelatin-coated 6 well plates. On day 2 cells were fed with differentiation media 1 with 5nM GSKiXV (Calbiochem, 361558) and 5µM IDE-1 (Tocris, 4015). On days 3, 4, and 5, cells were fed with differentiation media 2 [A-DMEM (GIBCO, 12491) supplemented with 2% B27 minus vitamin A, 1% Glutamax, 1% P/S] plus 5µM IDE-1. On day 6 cultures were split with 0.25% trypsin and plated at 3x105 cells/cm2 onto 804G conditioned medium-coated plates with 10µM Y-27632 in differentiation media 2. Cells were treated with 500nM A83-01 (Tocris, 2939) and 100nM LDN193189 (Selleck Chemicals, S2618).
Mouse Pluripotent Stem Cell Culture: All mouse PSC lines; AV3, p2lac40, Sox17GFP, Pax9Venus, were maintained on mouse embryonic fibroblasts in KO DMEM (GIBCO, 10829) supplemented with 15% Hyclone FBS (Thermo Scientific, SH30070.03), 1% Glutamax (GIBCO, 35050079), 1% NEAA (Cellgro, 25-025-CI), 100µM β-mercaptoethanol (Invitrogen, 21985023), 1x103 units/ml ESGRO LIF (Millipore, ESG1107). Cells were fed daily and split every 2-3 days with 0.25% trypsin (GIBCO, 25200114).
RNA was isolated using Trizol (Invitrogen, 15596-018) according to the manufacturer’s instructions.
500ng of total RNA was amplified and labeled using the Illumina Total Prep RNA Amplification Kit (Ambion, AMIL1791) according to the manufacturers’ instructions
Standard Illumina hybridization protocol
Standard Illumina scanning protocol
DE_Endoderm_3 replicate 3
Raw probe level intensity values were normalized using a variance stabilization approach by employing the R function vsn2 from the corresponding vsn package (Huber et al., 2002).