Mus musculus
Protocols: Mouse ESCs (mESCs) and gastruloids were maintained and generated as previously described (see Anlas, Baillie-Benson et al. 2021, Programmed Morphogenesis: Methods and Protocols, p131-147). In brief: mESCs were cultured in DMEM+serum/LIF, including passages every 2-3 days using TrypLE. For Gastruloid generation, mESCs were detached using TrypLE, cell counting was performed and the desired number of cells (usually ~300/well) were plated in 40ul N2B27 into all wells of a round bottom, low adhesion 96-well plate. After incubating for 48h, add 150ul N2B27+3uM CHIR99 (Wnt agonist). Samples were extracted from wells of low-adhesion, round-bottom 96-well plates into a 15ml centrifuge tube via a P1000 pipette with wide-bore RNAse-free filter tips or via organoid collection plates. Once all samples were collected, thus yielding one 15ml centrifuge tube per condition, aggregates were centrifuged at 180g for 2min. Hereafter, the supernatant was removed and 1ml TrypLE (ThermoFisher Scientific, 12604) was added per tube for dissociation and samples were incubated for 5min at (37C, 5% CO2). Then, TrypLE was deactivated using 4ml ESL and gastruloids were centrifuged for 2min at 180g. Supernatant was removed, samples were washed with 5ml cold (4C) 0.1% RNase-free BSA (ThermoFisher Scientific, AM2616) in DPBS−/− (BPBS) and centrifuged (2min, 180g). From this step onwards, samples were maintained at 4C. Following another washing step and removal of the supernatant, the pellet was resuspended in 50-100ul cold BPBS (depending on pellet size) by gently pipetting up and down 3-5x with a P200 tip. The cell suspension was then filtered through a 35um filtered cap tube (Corning, 352235). Cells were counted as previously described,aiming for a concentration of around 1,000,000 cells/ml. In case of high concentrations (above 1,500,000cells/ml) , the cell suspension was diluting with cold BPBS and re-counted. For the 0h timepoint, mESCs were trypsinized using 1ml Trypsin-EDTA 0.05%. Following neutralization (4ml ESL) and centrifugation (180g, 3min), the supernatant was discarded and cells were washed with 5ml cold BPBS. Cells were then kept on ice, until samples from other experimental conditions reached this washing step. At this point, all samples were joined together for centrifugation and further procedures illustrated above. cDNA libraries were constructed with Chromium Single Cell 3' GEM, Library & Gel Bead Kit v3 (10x Genomics) according to manufacturer's protocol.
ENA-FIRST-PUBLIC
2023-12-04T00:24:58Z
ENA-LAST-UPDATE
2023-12-04T00:24:58Z
External Id
SAMEA112711204
INSDC center name
EMBL
INSDC first public
2023-12-04T00:24:58Z
INSDC last update
2023-12-04T00:24:58Z
INSDC status
public
Submitter Id
E-MTAB-12749:Gastruloid_Rep2_24h
broker name
ArrayExpress
cell_line
ES E14
cell_type
embryonic stem cell
common name
house mouse
developmental stage
embryo
disease
normal
genotype
T/Bra::GFP
growth condition
organoid
isolate
not applicable
organism part
embryo
sample name
E-MTAB-12749:Gastruloid_Rep2_24h
scientific_name
Mus musculus
strain
129P2/Ola