W13093
Protocols: EpiSCs were seeded at ~6 x 10^3 cells/cm2 in 6-well cell-culture plates (Corning, 3506) pre-coated with 15 µg/ml human Fibronectin (Merck Millipore, FC01015), and differentiated to pre-somitic mesoderm (PSM) as described below. At desired time-points, cells were dissociated using Accutase (Thermo Fisher Scientific, 00-4555-56) for 3 min at 37°C and counted. 500,000 cells per condition were washed with ice-cold FACS buffer (10% (v/v) FBS in PBS) and resuspended into 1 ml ice-cold FACS buffer containing 1 µg/ml DAPI. Wild-type EpiSC lines were derived from E6.5 mouse embryos (129 background) and maintained in 6-well cell-culture plates (Corning, 3506) pre-coated with 15 µg/ml human Fibronectin (Merck Millipore, FC01015) in N2B27 medium (prepared as described in Ying Q.L. & Smith A.G., Methods Enzymol 365, 327-341 (2003)), supplemented with 20 ng/ml Activin A, 10 ng/ml bFGF and 1 µM XAV939 (EpiSC medium). All differentiation was conducted in serum-free, feeder-free and monolayer conditions in chemically-defined N2B27-based media. Culture conditions followed a modified protocol from Loh K.M. et al., Cell 166, 451-467 (2016). EpiSCs were seeded at ~6 x 10^3 cells/cm2 in 6-well cell-culture plates pre-coated with 15 µg/ml human Fibronectin, and cultured in EpiSC medium for 24 h. EpiSCs were first differentiated towards anterior primitive streak (APS) using 30 ng/ml Activin A, 4 µM CHIR-99021, 20 ng/ml bFGF and 100 nM PIK-90 (APS medium) for 12 h, and then to pre-somitic mesoderm (PSM) using 1 µM A-83-01, 3 µM CHIR-99021, 250 nM LDN193189 and 20 ng/ml bFGF (PSM medium) for 36 h. Single-cells were sorted into Eppendorf Polypropylene U-shaped 384-well Twin Tec PCR Microplates (Thermo Fisher Scientific, 10573035), containing 2 μl of lysis solution (0.2% (v/v) Triton X-100) supplemented with 0.4 U/μl RNasin Ribonuclease Inhibitor (Promega, N2515) and 400 nM indexed RT primer from group 1 (1-96 barcodes) or group 2 (97-192 barcodes) (Jaitin D.A. et al., Science, 343, 776-779 (2014)). Two wells were left empty in each plate, as a no-cell control during data analysis. Capture plates were prepared on the Bravo automated liquid handling robot station (Agilent) using 384-filtered tips (Agilent, 19133-142). Index sorting was performed using a FACS Aria III cell sorter (BD Biosciences) at the DanStem Flow Cytometry Platform (University of Copenhagen, Copenhagen, Denmark), sorting live cells as singlets. Immediately after sorting, plates were spun down, snap-frozen on dry ice and stored at -80°C until further processing. Semi-automated library preparation was performed as described in Jaitin D.A. et al., Science, 343, 776-779 (2014), using 10-12 total cycles of PCR amplification and AMPure XP beads (Beckman Coulter Life Sciences, A63881) for purification. DNA concentration was measured with a Qubit Fluorometer (Thermo Fisher Scientific, Q32854) and fragment size was determined with a Fragment analyzer (Agilent).
ENA first public
2021-07-21
ENA last update
2021-07-21
External Id
SAMEA7698603
INSDC center alias
Novo Nordisk Foundation Center for Stem Cell Biology (DanStem), University of Copenhagen
INSDC center name
Novo Nordisk Foundation Center for Stem Cell Biology (DanStem), University of Copenhagen
INSDC first public
2021-07-21T16:31:43Z
INSDC last update
2021-07-21T16:31:43Z
INSDC status
public
Submitter Id
E-MTAB-9774:W13093
broker name
ArrayExpress
cell line
129 EpiSC
cell type
epiblast cell
common name
house mouse
developmental stage
embryo
genotype
wild type genotype
organism part
inner cell mass
sample name
E-MTAB-9774:W13093
single cell well quality
OK