Protocols: cmPSC were encapsulated in agarose microgels and transferred into conventional tissue culture plates with or without mouse embryonic fibroblasts (MEFs) in differentiation permissive, serum-free N2B27 supplemented with 1% Matrigel, 2.5% Chemically Defined Lipids (Gibco) and 1% Penicillin-Streptomycin. Signalling inhibitors and activators were then added to this base medium: FGF (bFGF, 100 ng/ml, Cambridge Stem Cell Institute), CHIR (CHIR99021, 3 µM, Cambridge Stem Cell Institute), XAV (XAV939, 3 µM, Cell Guidance Systems SM38-200), ACTA (Activin A, 20 ng/ml, Cambridge Stem Cell Institute), SB43 (SB431542, 10 µM, Tocris 1614), BMP4 (BMP4, 20 ng/ml, R&D 314-BP). For each condition, a representative pool of 20-30 microgels were randomly selected for profiling by single-cell RNA sequencing. Structures were released from microgels by incubation with 1 U/ml Agarase (Thermo Fisher Scientific, EO0461) and dissociated for 10-15 minutes in 0.25% Trypsin-EDTA (Gibco, 25200056) followed by manual dissociation in N2B27 using a blunted microcapillary pipette. Single cells were isolated manually using a microcapillary pipette, transferred immediately into 4 µl single cell lysis buffer and snap frozen. RNA separation was performed using biotinylated oligo-dT30VN-tailed oligonucleotides (IDT) conjugated to Dynabeads Streptavidin C1 in an RNAse-inhibitor supplemented buffer solution. Sequencing libraries were prepared according to an adapted version of the G&T protocol previously described. Samples were transferred to a 96-well plate and prepared for both transcriptome and methylome sequencing, by physical separation of genomic DNA (gDNA) from messenger RNA (mRNA).
ENA first public
2022-06-15
ENA last update
2022-06-15
External Id
SAMEA10346788
INSDC center alias
UNIVERSITY OF CAMBRIDGE
INSDC center name
UNIVERSITY OF CAMBRIDGE
INSDC first public
2022-06-15T00:13:41Z
INSDC last update
2022-06-15T00:13:41Z
INSDC status
public
Submitter Id
E-MTAB-10639:BMP_MEF_J6
broker name
ArrayExpress
cell line
New4f
cell type
pluripotent stem cell
common name
white-tufted-ear marmoset
developmental stage
embryo
disease
normal
genotype
wild type genotype
growth condition
coculture with mouse embryonic fibroblasts
organism part
inner cell mass
sample name
E-MTAB-10639:BMP_MEF_J6