organism: Callithrix jacchus
description: Protocols: Bulk whole genome bisulfite sequencing was conducted on three replicates of cmTSC, primed cmPSCs, and naïve cmPSCs. Marmoset PSCs were maintained in KSR/bFGF medium, which is comprised of Dulbecco's modified Eagle medium (DMEM)/F12 (21331, Gibco) supplemented with 20% Knockout Serum Replacement (KSR) (10828028, Thermo Fisher Scientific), 1% GlutaMAX (35050061, Thermo Fisher Scientific), 1% MEM non-essential amino acids (11140035, Thermo Fisher Scientific), 100 μM β-mercaptoethanol (21985023, Thermo Fisher Scientific) and 10 ng/mL bFGF (Cambridge Stem Cell Institute). Cells were routinely cultured on mitomycin C (M4287, Sigma-Aldrich) inactivated mouse embryonic fibroblast (MEF) feeder cells (Cambridge Stem Cell Institute) under 5% O2 and 5% CO2 at 37 °C. The medium was changed daily, and cells were passaged every 2–4 days by dissociation with Accutase (00-4555-56, Thermo Fisher Scientific) for 5 min. Marmoset PSCs were reset chemically using as previously described (Bergmann et al., 2022a). In short, conventional marmoset PSCs were seeded as clumps of 2–5 cells one day before resetting at 50,000 cells per well of a 12-well plate (1.3 × 104 cells per cm2) on MEFs. After 24 h, the medium was changed to PLAXA, which comprised N2B27 medium (NDiff, Y40002, Takara Bio) supplemented with 1% chemically defined lipids, 1 μM PD0325901 (Cambridge Stem Cell Institute), 10 ng/ml recombinant human LIF (Cambridge Stem Cell Institute), 50 μg/ml 1 L-ascorbic acid (Sigma-Aldrich), 2 μM XAV939 (SM38-200, Cell Guidance Systems) and 20 ng/ml activin A (Cambridge Stem Cell Institute). Throughout conversion, cells were passaged with Accutase (00-4555-56, Thermo Fisher Scientific) 1:1.5 every 3–4 days. Dome-shaped colonies first emerge at day 4–5 and naïve conversion is complete by day 9. Naïve PSCs were prepared as previously described on MEFs and cultured to 60-70% confluency. Naïve PSC media was removed and PSCs were washed in PBS. Media was replaced with indicated culture conditions and differentiated for 5 days. OKAE conditions were as previously described (Okae et al., 2018). Other conditions include OK XAV PD: advanced DMEM/F12 supplemented with 55 μM β-mercaptoethanol, 0.3% BSA, 1% ITS-X supplement, 50 ng/ml EGF, 2 μM XAV939, 1 μM PD0325901, 1 mM A83-01, 1 mM SB431542, 0.7 mM VPA and 1 mM Y27632 and PAVS: advanced DMEM/F12 supplemented with 0.3% BSA, 1% ITS-X supplement, 55 μM β-mercaptoethanol, 1% chemically defined lipids, 1x GlutaMAX (Thermo Fischer), 1 μM PD0325901, 1 mM A83-01, 1 mM SB431542, 0.7 mM VPA and 1 mM Y27632. Both conditions were cultured on inactive MEFS unless otherwise specified. 5µg/ml collagen IV coating was generated by resuspending collagen in PBS and coating at 37°C for 2 hours. Additional molecules added during screening includes 2 μM XAV939, 20ng/mL BMP4, 0.5μM LDN193189 (LDN), 20ng/mL Activin A, 100ng/mL FGF2, 2 µM CHIR99021 (CHIR), 3 μM IWP2, 20 μM forskolin, 50 ng/ml EGF, 50 ng/mL HGF, 2 mM A83-01, and 2.5 µM PGE2. Media was changed every 24 hours. After 4-5 days or at 90% confluency, epithelial colonies were washed with PBS and dissociated with TrypLE for 6-8 minutes or until cells balled up and formed 2-3 cell clumps. TrypLE was carefully removed to not disturb cells and cells were resuspended and replated in culture media supplemented with 10 µM Y-27 for 24 hours. cmTSCs were passaged 1:2 every 7 days or until 80% confluence was reached. Cells were cultured in 5% CO2 and 5% O2 at 37°C. Genomic DNA was extracted using a Monarch Genomic DNA Purification Kit (NEB). Standard library construction protocols were used.
identifiers.namespace: BioSample
identifiers.id: SAMEA112229862
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2023-11-21T00:50:42Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-11-21T00:50:42Z
attributes.tag: External Id
attributes.value: SAMEA112229862
attributes.tag: INSDC center name
attributes.value: University of Cambridge, Department of Physiology, Development and Neuroscience
attributes.tag: INSDC first public
attributes.value: 2023-11-21T00:50:42Z
attributes.tag: INSDC last update
attributes.value: 2023-11-21T00:50:42Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-12453:Naive rep 1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_type
attributes.value: pluripotent stem cell
attributes.tag: common name
attributes.value: white-tufted-ear marmoset
attributes.tag: developmental stage
attributes.value: embryo
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: inner cell mass
attributes.tag: phenotype
attributes.value: naive
attributes.tag: sample name
attributes.value: E-MTAB-12453:Naive rep 1
attributes.tag: scientific_name
attributes.value: Callithrix jacchus