11422_N702_S502
Mus musculus
Protocols: Cells were dissociated with Accutase (Millipore). ESCs and iPSCs were cultured in N2B27 medium supplemented with MEK and GSK3 inhibitors (2i) and leukaemia inhibitory factor (2iLIF). EpiSCs were cultured in N2B27 supplemented with FGF2, ActivinA and XAV939 (FA). N2B27 medium comprised 1:1 DMEM/F-12 and Neurobasal (Gibco), 2 mM L-glutamine (Gibco), 1x penicillin-streptomycin (Sigma), 0.1 mM 2-mercaptoethanol (Gibco), 1% B27 (Gibco) and 0.5% N2 (homemade). As required, N2B27 was supplemented with 20 ng/ml murine LIF (Hyvonen lab, Cambridge), 3 μM CHIR99021 and 1 μM PD0325901 (Stewart lab, Dresden), 12.5 ng/ml FGF2 and 20 ng/ml ActivinA (Hyvonen lab, Cambridge), 6.25 µg/ml XAV939 (Tocris), 3 µM DMH2 (Tocris), or 0.6 µM LDN193189 (Sigma). For ESCs and iPSCs, culture vessels were coated with 0.15% gelatin (Sigma) in PBS (Sigma) and incubated in 7% CO2. For EpiSC culture and reprogramming experiments, tissue-culture flasks were coated with 10 µg/ml fibronectin (Millipore) in PBS (Sigma) and incubated in 7% CO2 and 5% O2. ESCs, iPSCs and EpiSCs were dissociated with accutase (Millipore) during passaging. For selected experiments, ESCs were cultured in serum+LIF medium containing GMEM (Sigma), 10% fetal bovine serum (Gibco), 1x non-essential amino acids (Gibco), 1 mM sodium pyruvate (Sigma), 2 mM L-glutamine (Gibco), 1x penicillin-streptomycin (Sigma), 0.1 mM 2-mercaptoethanol (Gibco). 20 ng/ml murine LIF (Hyvonen lab, Cambridge) and 10ng/ml BMP4 (Miltenyi Biotec) was supplemented as indicated. iEssrb; iPStat3 and iKlf2 indicates reprogrammed iPS cells that are derivatives of the parental line. This study also includes samples that were flow sorted on the Rex1-GFP reporter. All indicates an unselected sample whilst low is a flow sorted subset with low GFP expression - the Rex1-GFP reporter is used as a proxy for naive pluripotency to assess the degree of reprogramming in a time series experiment. EvrtTa3 is an abbreviation for EmptyVector+CAG.rtTA3 - a negative control incorporated into parental EpiSCs. GY118F is a transgenic receptor introduced by piggyBac transformation. This is a transposon system that confers stable integration of a cassette at random sites in the genome, rather than a locus-specific knock-in. EpiSCs were plated in FA without selection at a density of 2000 cells/24-well or equivalent. For siRNA experiments, 10000/24-well or equivalent was used instead to compensate for transfection toxicity. The following day, reprogramming was induced by medium change to 2iLIF or subset components thereof as indicated. GY118F transgenic receptor was stimulated with 30 ng/ml human GCSF (Peprotech), whereas expression of other transgenes was induced with 1 µg/ml doxycycline (MP Biomedicals). After 4 days, transgene induction was withdrawn and 20 μg/ml blasticidin (Gibco) was applied to select for Rex1::dGFP.IRES.bsd reporter activity. Cells were deposited individually by flow cytometry (Influx 5, BD Biosciences) into 96-well plates containing lysis buffer. Cells were sorted on days 2, 3 and 4 by flow cytometry (Influx 5, BD Biosciences) and deposited individually into 96-well plates containing lysis buffer. dGFP+ DAPI- cells were selected for all reprogramming intermediates, ESC and iPSC samples. dGFP- DAPI- cells were sorted from EpiSC cultures. Sequencing libraries were prepared according to the Smart-seq2 protocol (Picelli et al., Nat Protoc. 9:171-81) using the Nextera XT DNA Library Prep Kit (Illumina).
ENA-FIRST-PUBLIC
2019-06-30T04:02:40Z
ENA-LAST-UPDATE
2019-06-07T15:14:22Z
External Id
SAMEA5700336
INSDC center name
UNIVERSITY OF CAMBRIDGE
INSDC first public
2019-06-30T04:02:40Z
INSDC last update
2019-06-07T15:14:22Z
INSDC status
public
Submitter Id
E-MTAB-7901:11422_N702_S502
broker name
ArrayExpress
cell line
Rex1::dGFP
cell type
induced pluripotent stem cell
common name
house mouse
developmental stage
embryo stage
disease
normal
genotype
Rex1-GFP; Esrrb transgene
organism part
epiblast
phenotype
not available
post analysis well quality
pass
protocol
Esrrb reprogramming
sample name
E-MTAB-7901:11422_N702_S502
scientific_name
Mus musculus
single cell quality
OK
single cell well quality
OK
strain
129/Ola