Sample 31
Mus musculus
Protocols: Mouse ESCs were maintained without feeders in “LIF+serum” medium (DMEM (high glucose, no glutamine, with sodium bicarbonate, Invitrogen) supplemented with 15% ES-qualified EmbryoMax Fetal Calf Serum (Millipore), 10 ng/ml murine LIF (EMBL Protein Expression and Purification Core Facility), 1x Non-Essential Amino Acids, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin and 100 μg/ml strep- tomycin, 0.1 mM 2-mercaptoethanol (all Invitrogen) on culture dishes (Nunc) coated with 0.1% gelatin (Sigma) solution and cultured at 37◦C with 5% CO2. Medium was changed daily and cells were passaged every other day with 0.05% Trypsin-EDTA (Invitrogen) at a passaging ratio of 1/6- 1/12. N2B27 medium was prepared from a 1:1 mixture of DMEM/F12 (without HEPES, with L-glutamine) and neurobasal medium with 0.5x B-27 (without vitamin A) and 0.5x N-2 supplements, 100 U/ml penicillin and 100 μg/ml streptomycin, 0.25 mM L-glutamine, 0.1 mM 2-mercaptoethanol (all Invitrogen), 10 μg/ml BSA fraction V and 10 μg/ml human recombinant insulin (both Sigma). mESCs were differentiated towards an endodermal progenitor fate following the method described by Borowiak et al. (cell Stem Cell, 2009). Briefly, mESCs were seeded at a density of 2500 cells per cm2 onto 0.1% gelatin coated dishes one day prior to the start of the differentiation procedure. The following day, cells were rinsed in D-PBS and switched to endodermal differentiation medium (Advanced RPMI 1640 (ThermoFisher), 1 μM IDE-1 (Tocris), 0.2% (v/v) fetal calf serum (Millipore), 2 mM L-glutamine (Sigma)). Samples collected 24 h after switching to the differentiation regime are referred to as “day 1” differentiation samples. Medium was replaced every other day. mESCs were differentiated towards a mesodermal progenitor fate following the protocol described in Torres et al. (PLoS ONE, 2012). Briefly, mESCs were seeded at a density of 2500 cells per cm2 onto 0.1% gelatin coated dishes one day prior to the start of the differentiation procedure. The following day, cells were rinsed in D-PBS and switched to mesodermal differentiation medium (Glasgow's MEM (ThermoFisher), 10% (v/v) KnockOut Serum Replacement (ThermoFisher), 0.1 mM 2-mercaptoethanol (Invitrogen), 1x non-essential amino acids (Gibco) and 1 mM sodium pyruvate (Gibco)). Medium was replaced every other day. mESCs were differentiated towards an ectodermal progenitor fate following the protocol developed by Ying et al. (Nature Biotechnology 2003). Briefly, mESCs were seeded at a density of 7500 cells per cm2 onto 0.1% gelatin coated dishes one day prior to the start of the differentiation procedure. The following day, cells were washed in D-PBS and switched to N2B27 medium. all-trans-Retinoic acid (Sigma) was added at 1 μM to the differentiation medium 24h after the start of the differentiation procedure. Medium was replaced every three to four days. mESCs were differentiated towards an Epiblast Stem Cell like state by culture in N2B27 medium supplemented with 12 ng/ml murine FGF2 and 20 ng/ml murine Activin A (both PeproTech). Medium was changed daily and cells were passaged every other day with StemPro Accutase (Invitrogen) at a passaging ratio of 1/3–1/6. Barcoded mRNA libraries were prepared using TruSeq RNA Sample Preparation (Illumina) following the manufacturer's instructions.
ENA first public
2019-12-06
ENA last update
2016-07-18
External Id
SAMEA4346667
INSDC center alias
EMBL
INSDC center name
European Molecular Biology Laboratory
INSDC first public
2019-12-06T04:04:12Z
INSDC last update
2016-07-18T17:40:21Z
INSDC status
public
Submitter Id
E-MTAB-4904:Sample 31
broker name
ArrayExpress
cell type
embryonic stem cell
common name
house mouse
developmental stage
5 day after differentiation
growth condition
N2B27 Activin+FGF EpiSC differentiation
sample name
E-MTAB-4904:Sample 31