ERS3734383 SAMEA5945916 9606 Homo sapiens Protocols: Man5 human ESCs (http://dx.doi.org/10.1016/j.stemcr.2016.07.006, https://doi.org/10.1016/j.stemcr.2018.09.013) were thawed and maintained on mitotically inactivated MEFs in Knock-Out DMEM media supplemented with 20% Knock-Out Serum Replacement, 1% Minimum Essential Medium (MEM) Non-Essential Amino acid, 2mM L-glutamine, 25U/ml penicillin-streptomycin, 0.1mM 2-mercaptoethanol and 8ng/ml human recombinant bFGF. Before differentiation, hESCs were feeder-depleted by culturing on Geltrex for 4-5 days in TeSR-E8 media supplemented with 25U/ml Pen/Strep. To generate EBs, hESCs were treated with EDTA and gently dissociated with StemPro EZPassage Cell Passaging Tool. The generated clumps were resuspended in EB media, containing IMDM, 20% FCS, 2mM L-gln, 25U/ml Pen/Strep, 150ug/ml Transferrin, 50ug/ml Ascorbic Acid, 4.5x10-4 M monothioglycerol, Geltrex (1:200) and 10uM ROCK inhibitor. Clumps were centrifuged and resuspended in aggregation medium, constituted by StemPro-34 SFM supplemented with 2mM L-gln, 50U/ml Pen/Strep, 150ug/ml Transferrin, 50ug/ml Ascorbic Acid, 4.5x10-4 M MTG, Geltrex (1:200), 10uM ROCK inhibitor, 10ng/ml BMP4. These were plated in low-attachment dishes and incubated at 37°C 5% O2 5% CO2. After 24 hours, 5ng/ml bFGF was added to each dish. On the second day of culture, EBs were collected, decanted by gravity and resuspended in Induction 1 medium: 2mM L-gln, 50U/ml Pen/Strep, 150ug/ml Transferrin, 50ug/ml Ascorbic Acid, 4.5x10-4 M MTG, 10ng/ml BMP4, 5ng/ml bFGF, 0.9ng/ml Activin A in StemPro-34. At day 4, EBs were collected, decanted by gravity and resuspended in Induction 2 medium: 2mM L-gln, 50U/ml Pen/Strep, 150ug/ml Transferrin, 50ug/ml Ascorbic Acid, 4.5x10-4 M MTG, 5ng/ml bFGF, 12ng/ml VEGF in StemPro-34. The CD144+CD31+ population was isolated at day 6 of EB differentiation by FACS. Used 10X sample collection protocol, detailed in CG000053_CellPrepGuide_RevC.pdf Used 10X sample collection protocol, detailed in CG000053_CellPrepGuide_RevC.pdf Used 10X sample collection protocol, detailed in CG000053_CellPrepGuide_RevC.pdf ENA-FIRST-PUBLIC 2020-05-02T04:03:47Z ENA-LAST-UPDATE 2019-09-09T15:08:44Z External Id SAMEA5945916 INSDC center name Bioinformatics Core Facility, Developmental Haematopoiesis Group, Faculty of Biology, Medicine and Health, The University of Manchester INSDC first public 2020-05-02T04:03:47Z INSDC last update 2019-09-09T15:08:44Z INSDC status public Submitter Id E-MTAB-8271:Sample 1 broker name ArrayExpress cell line MAN5 cell type haemogenic endothelium cell common name human progenitor cell type embryonic stem cell sample name E-MTAB-8271:Sample 1 scientific_name Homo sapiens