REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rabbit polyclonal anti-AKT2 (phospho S474), (used in gastruloid trajectory)	Abcam	Cat # ab38513; RRID: AB_867564
Rabbit monoclonal anti-Activin A Receptor Type IB/ALK-4, (used in gastruloid trajectory)	Abcam	Cat # ab109300; RRID: AB_10860328
Mouse monoclonal anti-β-catenin, (used in gastruloid trajectory)	BD Biosciences	Cat # 610154; RRID: AB_397555
Rabbit monoclonal anti-Brachyury, (used in gastruloid trajectory)	Abcam	Cat # ab209665; RRID: AB_2750925
Rabbit monoclonal anti-Cdx2, (used in gastruloid trajectory)	Abcam	Cat # ab76541; RRID: AB_1523334
Goat polyclonal anti-Dppa4	R&D Systems	Cat # AF3730; RRID: AB_2094166
Mouse monoclonal anti-E-cadherin, (used in gastruloid trajectory)	BD Biosciences	Cat # 610181; RRID: AB_397580
Rabbit monoclonal anti-EGFR (phospho Y1068), (used in gastruloid trajectory)	Abcam	Cat # ab40815; RRID: AB_732110
Rabbit polyclonal anti-Fibronectin, (used in gastruloid trajectory)	Merck	Cat # F3648; RRID: AB_476976
Rabbit monoclonal anti-FoxA2, (used in gastruloid trajectory)	Abcam	Cat # ab108422; RRID: AB_11157157
Rabbit monoclonal anti-Gata6, (used in gastruloid trajectory)	Cell Signaling Technology	Cat # 5851S; RRID: AB_10705521
Rabbit monoclonal anti-Hes1, (used in gastruloid trajectory)	Cell Signaling Technology	Cat # 11988S; RRID: AB_2728766
Rabbit monoclonal anti-Ki67, (used in gastruloid trajectory)	Abcam	Cat # ab16667; RRID: AB_302459
Mouse monoclonal anti-N-cadherin, (used in gastruloid trajectory)	BD Biosciences	Cat # 610920; RRID: AB_2077527
Rabbit polyclonal anti-Nanog	Abcam	Cat # ab80892; RRID: AB_2150114
Mouse monoclonal anti-Nestin	Millipore	Cat # MAB353; RRID: AB_94911
Mouse monoclonal anti-Oct3/4, (used in gastruloid trajectory)	BD Biosciences	Cat # 611203; RRID: AB_398737
Rabbit polyclonal anti-Otx1/2	Abcam	Cat # ab21990; RRID: AB_776930
Goat polyclonal anti-Otx2	RnD	Cat# AF1979; RRID:AB_2157172
Mouse monoclonal anti-p44/42 MAPK (Erk1/2), (used in gastruloid trajectory)	Cell Signaling Technology	Cat # 4696S; RRID: AB_390780
Rabbit monoclonal anti-Pax6	Abcam	Cat# ab195045; RRID:AB_2750924
Rabbit polyclonal anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), (used in gastruloid trajectory)	Cell Signaling Technology	Cat # 9101S; RRID: AB_331646
Rabbit polyclonal anti-Phospho-NPM (Thr95), (used in gastruloid trajectory)	Cell Signaling Technology	Cat # 3517S; RRID: AB_2155177
Rabbit polyclonal anti-Phospho-Smad2 (Ser465/467), (used in gastruloid trajectory)	Thermo Fisher Scientific	Cat # 44-244G; RRID: AB_2533614
Goat polyclonal anti-Sox1, (used in gastruloid trajectory)	Cell Signaling Technology	Cat # 4194; RRID: AB_1904140
Rabbit monoclonal anti-Sox2	Cell Signaling Technology	Cat # 23064S; RRID: AB_2714146
Rat monoclonal anti-Sox2, (used in gastruloid trajectory)	Thermo Fisher Scientific	Cat # 14-9811-82; RRID: AB_11219471
Rabbit monoclonal anti-Tbr2/Eomes, (used in gastruloid trajectory)	Abcam	Cat # ab183991; RRID: AB_2721040
Mouse monoclonal anti-Tubulin beta-3	BioLegend	Cat# 801201; RRID:AB_2313773
Donkey anti-rabbit Alexa Fluor 488	Thermo Fisher Scientific	Cat # A-21206; RRID:AB_2535792
Donkey anti-rabbit Alexa Fluor 488 Plus	Thermo Fisher Scientific	Cat # A32790; RRID:AB_2762833
Donkey anti-rat Alexa Fluor 488	Thermo Fisher Scientific	Cat # A-21208; RRID:AB_2535794
Donkey anti-rabbit Alexa Fluor 568	Thermo Fisher Scientific	Cat # A10042; RRID:AB_2534017
Donkey anti-mouse Alexa Fluor 568	Thermo Fisher Scientific	Cat # A10037; RRID:AB_2534013
Donkey anti-rabbit Alexa Fluor 647	Thermo Fisher Scientific	Cat # A-31573; RRID:AB_2536183
Donkey anti-rabbit Alexa Fluor 647 Plus	Thermo Fisher Scientific	Cat # A32795; RRID:AB_2762835
Donkey anti-mouse Alexa Fluor 647	Thermo Fisher Scientific	Cat A-31571; RRID:AB_162542
Donkey anti-goat Alexa Fluor 647	Thermo Fisher Scientific	Cat # A-21447; RRID:AB_2535864
Donkey anti-goat Alexa Fluor 647	Abcam	Cat # ab150135; RRID:AB_2687955
Chemicals, peptides, and recombinant proteins
CHIR99021	STEMCELL Technologies	Cat # 72054
PD0325901	STEMCELL Technologies	Cat # 72182
Mouse Recombinant LIF	STEMCELL Technologies	Cat # 78056
SB431542	Tocris	Cat # 1614
BMS605541	Tocris	Cat # 6069
IWP2	Tocris	Cat # 3533
SU5402	STEMCELL Technologies	Cat # 73914
XAV939	STEMCELL Technologies	Cat # 72672
Screening library (see Table S7)	Novartis	N/A
PD161570	Novartis	N/A
Sorafenib	Novartis	N/A
RGD-(Arg-Gly-Asp)-peptide	Selleck Chemicals	Cat # S8008
Focal Adhesion Kinase Inhibitor III	Merk	Cat # 5.04045
Deposited data
Single cell RNA sequencing and multiome data	This paper	GEO: GSE229513
Bulk RNA-sequencing data	This paper	GEO: GSE229386
Experimental model/ Cell lines
BramCh/Sox2Ve T::H2B-mCherry,Sox2::H2B-Venus	Laboratory of Jesse V. Veenvliet	N/A
CGR8	Laboratory of Matthias Lutolf	129P2
E14	Laboratory of Matthias Lutolf	129P2
Mesp1-GFP	Laboratory of Matthias Lutolf, originating from: Laboratory of Cédric Blanpain	N/A
miR-290-mCherry/mir-302-eGFP, DRC	Laboratory of Matthias Lutolf, originating from the Laboratory of Robert Blelloch	N/A
Sox1-GFP::Brachyury-mCherry (SBR)	Laboratory of Matthias Lutolf originating from: Laboratory of David Suter	based on CGR8 129P2
129sv/ev	Experiments using this cell line were performed in the Laboratory of Denis Duboule.	CMTI-1, Embryomax
Oligonucleotides
AllStars Negative Control siRNA	Qiagen	Cat #SI03650318
Mm_Sox2_4 FlexiTube siRNA	Qiagen	Cat #SI01429596
Mm_Sox2_3 FlexiTube siRNA	Qiagen	Cat #SI01429589
Mm_Fn1_1 FlexiTube siRNA	Qiagen	Cat #SI01004059
Mm_Fn1_2 FlexiTube siRNA	Qiagen	Cat # 1027415, SI01004066
Software and algorithms
Fiji/ImageJ	N/A	https://imagej.net/Fiji
FlowJo™	BD Life Sciences	https://www.flowjo.com
Python	Python Software Foundation	https://www.python.org
R	R Project	https://www.r-project.org
Gastruloid feature extraction pipeline	This paper https://github.com/fmi-basel/gliberal-gastruloid-2023-methods	https://doi.org/10.5281/zenodo.7858557
Other
384-well black/clear round bottom ultra-low attachment spheroid microplates	Corning	Cat # 4516
EL406 washer dispenser	BioTek Instruments	N/A
CyBio SELMA 384/25 μl	Analytik Jena AG	N/A
Integra Assist Plus	Integra	N/A