2025-06-06
ENA first public
ERC000011
ENA-CHECKLIST
SAMEA114610472
External Id
EMBL
INSDC center name
2023-11-14T14:29:15Z
INSDC last update
public
INSDC status
ERS16973739
SRA accession
E-MTAB-13543:48c2
Submitter Id
ArrayExpress
broker name
E14, Bra::GFP mESCs
cell_line
not collected
collection date
house mouse
common name
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) with the exception that no CHIR99 pulse (Wnt agonist) was applied at 48h post-aggregation. 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 300 cells/well were plated in 40µl N2B27 into all wells of a round bottom, low adhesion 96-well plate. At 24h after aggregation, 40�l N2B27 (control) or 40µl N2B27 containing 12mM 2-deoxy-D-glucose (2-DG treated, final concentration: 6mM 2-DG) were added. At 48 h after aggregation, 2-DG was washed out by adding 150μL N2B27 containing, removing 180µl medium, and adding again 150μL pre-warmed N2B27 containing. At 72 h after aggregation, 140µl medium were removed and 150μL fresh N2B27 added. Gastruloids were collected at 48h and 96h post-aggregation for RNA extraction. RNA was extracted from single gastruloids using the RNeasy Micro Kit (QIAGEN, #74004). RNA quality control was performed with the Agilent RNA 6000 Pico Kit (Agilent, #5067-1513) on the Bioanalyzer system (Agilent). Sequencing libraries were prepared following the SmartSeq2 protocol described by Picelli et al. (Nat Protoc 2014).
description
dma:collection:36374
dm app
not collected
geographic location (country and/or sea)
post-aggregation
initial time point
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
gastruloid
organism part
embryonic stem cell
progenitor cell type
Mus musculus
scientific_name
48c2
title