characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-01-01T00:28:40Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-01-01T00:28:40Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA111348804
characteristics.characteristic: External Id
characteristics.text: Universitat Pompeu Fabra
characteristics.characteristic: INSDC center name
characteristics.text: 2023-01-01T00:28:40Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-01-01T00:28:40Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS13446351
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12148:Sample 172
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: embryonic stem cell
characteristics.characteristic: cell_type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: At 120h, 144h, 168h, 192h and 216h, single cells from 2 independent gastruloid cultures were sorted on a FacsAriaII instrument and deposited into half 96-well plates, 1 plate per time-point and sorting gate. Cells were stained with C-Kit-APC-Cy7 (clone 2B8, Biolegend), CD41-PE (MWReg30, Biolegend), CD45-APC (clone 30-F11, Biolegend) and Sca1-PE-Cy7 (clone D7, Biolegend), and CD41+ cells and C-Kit+Sca1+ cells were sorted at 144h, CD45+ cells and C-Kit+Sca1+ cells at 192h, and CD45+ cells sorted at 216h. At 120h, 144h, 168h, and 192h, bulk individual cells were also sorted on a live singlets gate, for a total of 9 plates (4 bulk, 5 fluorescence-sorted). Cells were deposited into a lysis buffer as per the published Smart-Seq2 sequencing protocol, centrifuged and snap-frozen. Flk1-GFP mouse ES cells were cultured in 3-dimensional gastruloid aggregates in the presence of mesodermal and hemogenic-promoting cytokines for 216h. Flk-1 mouse embryonic stem cells (mES) were cultured in ESLIF medium in gelatinised flasks and treated in 2iLIF 24 hours prior to gastruloid assembly. 250 mES cells were seeded in each well of a U-bottom, cell-repellent 96-well plate. After 48 hours, N2B27 medium supplemented with 100 ng/ml Activin A and 3 μM chiron was added. At 72 h, N2B27 with 5 ng/ml of Vegf and Fgf2 each was replaced. From 72 h to 144 h, each day medium was removed and replaced with N2B27 + Vegf + Fgf2. At 144 h, the medium was further supplemented with Shh at 20 ng/ml. From 168 h to 216 h, the medium was N2B27 + 5 ng/ml Vegf, plus 20 ng/ml mTpo, 100 ng/ml mFlt3l, and 100 ng/ml mScf. Cell lysis with 0.2% Triton X-100, 1U/µl RNase inhibitor, Thermo Fisher Scientific Reverse transcription was performed using SuperScript II (Thermo Fisher Scientific) in the presence of 1 μM oligo-dT30VN (IDT), 1 μM Template-switching oligonucleotides (QIAGEN), and 1 M betaine. cDNA was amplified using the KAPA Hifi Hotstart ReadyMix (Kapa Biosystems) and IS PCR primer (IDT), with 22 cycles of amplification. Following purification with Agencourt Ampure XP beads (Beckmann Coulter), product size distribution and quantity were assessed on a Bioanalyzer using a High Sensitivity DNA Kit (Agilent Technologies). A total of 140 pg of the amplified cDNA was fragmented using Nextera XT (Illumina) and amplified with with double indexed Nextera PCR primers (IDT). Products of each well of the 96-well plate were pooled and purified twice with Agencourt Ampure XP beads (Beckmann Coulter). Final libraries were quantified and checked for fragment size distribution using a Bioanalyzer High Sensitivity DNA Kit (Agilent Technologies).
characteristics.characteristic: description
characteristics.text: embryo stage
characteristics.characteristic: developmental stage
characteristics.text: normal
characteristics.characteristic: disease
characteristics.text: Flk1-GFP
characteristics.characteristic: genotype
characteristics.text: gastruloid differentiation
characteristics.characteristic: growth condition
characteristics.text: CD45+
characteristics.characteristic: immunophenotype
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: embryoid body
characteristics.characteristic: organism part
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: Sample 172
characteristics.characteristic: title