organism: Mus musculus
description: 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).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2023-01-01
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-01-01
attributes.tag: External Id
attributes.value: SAMEA111348719
attributes.tag: INSDC center alias
attributes.value: Universitat Pompeu Fabra
attributes.tag: INSDC center name
attributes.value: Universitat Pompeu Fabra
attributes.tag: INSDC first public
attributes.value: 2023-01-01T00:28:40Z
attributes.tag: INSDC last update
attributes.value: 2023-01-01T00:28:40Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-12148:Sample 666
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_type
attributes.value: embryonic stem cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: embryo stage
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: Flk1-GFP
attributes.tag: growth condition
attributes.value: gastruloid differentiation
attributes.tag: immunophenotype
attributes.value: CD41+
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: embryoid body
attributes.tag: sample name
attributes.value: E-MTAB-12148:Sample 666