title: Single-gastruloid trancriptomics: control vs glycolysis inhibited at 48h and 96h post-aggregation
design: Single-gastruloid trancriptomics: control vs glycolysis inhibited at 48h and 96h post-aggregation
library_construction_protocol: 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).