Nature - USA (2020-01-23)

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nature research | reporting summary

October 2018

Field-specific reporting

Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences

For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design

All studies must disclose on these points even when the disclosure is negative.

Sample size No statistical methods were used to predetermine sample size. The number of embryos used in each experiment were provided in methods

and figure legends. For these experiments with some variations, we examined at least 3. For these experiments with highly consistent data,

we used at least two embryos because of limited embryos. Fir RNA-Seq, the sample size was determined when the main cell lineages at each

developmental stages were captured. Related statistical analysis provides the rationale for sufficiency of the sample sizes.

Data exclusions Two scRNA-seq profiles whose numbers of genes with abundance levels more than 1 FPKM were smaller than 2000 were excluded. The

criteria was established in the analysis procedure when we found that the two scRNA-seq profiles with < 2000 expressed genes were isolated

far away from other scRNA-seq profiles.

Replication Methods and figure legends indicated the exact number of embryos replicated in each experiment. All attempts at replication were

successful.

Randomization The embryos used in each experiment were chosen at random. The experiments were not randomized.

Blinding The investigators were not blinded to allocation during experiments and outcome assessment. Data collection and analysis were performed

by different people, the sample classification were replaced by simple marks during data analysis.

Reporting for specific materials, systems and methods

We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material,

system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.

Materials & experimental systems

n/a Involved in the study

Antibodies

Eukaryotic cell lines

Palaeontology

Animals and other organisms

Human research participants

Clinical data

Methods

n/a Involved in the study

ChIP-seq

Flow cytometry

MRI-based neuroimaging

Antibodies

Antibodies used Immunostaining:

mouse anti-OCT3/4 (Santa Cruz, SC5279,C-10, H1612 1:400), rabbit anti-Brachyury (T) (Santa Cruz, SC20109, polyclonal, A0616,

1:50), rabbit anti-SOX2 (Millipore, AB5603, polyclonal, 2826070, 1:400), goat anti-SOX17 (R&D Systems, AF1924, polyclonal,

KGA0815042,1:250), rabbit anti-KLF4 (Millipore, 09-821, polyclonal, 2383578, 1:400), rabbit anti-KLF17 (Atlas Antibodies,

HPA024629, polyclonal, C117502, 1:250), rabbit anti-β-Catenin (Abcam, AB32572, E247, GR184212-37, 1:300), mouse anti-E-

cadherin (Abcam, AB76055, M168, GR299147-4, 1:100), rabbit anti-Cytokeratin 7 (CK7) (Abcam, AB181598, EPR17078,

GR3214132-10, 1:300), rabbit anti-N-cadherin (Abcam, AB12221, polyclonal, 40975, 1:200), mouse anti-OTX2 (Santa Cruz,

SC514195, D-8, G0816, 1:100), goat anti-LEFTY1 (R&D Systems, AF746, polyclonal, CMM0111101, 1:100), goat anti-SOX1 (R&D

Systems, AF3369, polyclonal, XUV0618081, 1:500), rabbit anti-PAX6 (Biolegend, 901301, Poly 19013, b267205, 1:500), rabbit

anti-Laminin (Sigma-Aldrich, L9393, polyclonal, 028M4890V, 1:50), rabbit anti-TEAD4 (Atlas Antibodies, HPA056896, polyclonal,

R78063, 1:150), goat anti-FOXA2 (Santa Cruz, SC6554, polyclonal, D1216, 1:100), goat anti-OCT3/4 (Santa Cruz, SC8628,

polyclonal, G3201, 1:250), Mouse anti-hCG (Abcam, AB9582, 5H4-E2, GR308272-2, 1:100), rabbit anti-PRDM14 (Millipore,

AB4350, polyclonal, 2897240, 1:50), goat anti-TFCP2L1 (R&D Systems, AF5726, polyclonal, CCUG0115021,1:200), goat anti-

NANOG (R&D Systems, AF1997, polyclonal, KKJ0514091, 1:250), mouse anti-PODXL (R&D Systems, MAB1658, 222328,

JKW0218041, 1:400), mouse anti-HLA-G (Abcam, AB52455, 4H84, GR251679-19, 1:200), goat anti-GATA6 (R&D Systems,

AF1700, polyclonal, KWT0419021, 1:200), mouse anti EZRIN (Sigma-Aldrich, E8897, 3C12, 117M4875V, 1:500), rabbit anti-CER1

(Sigma-Aldrich, HPA019917, polyclonal, R10176, 1:50), Phalloidin (F-actin) Alexa Fluor®488 (Thermo Fisher Scientific, A12379,

direct labeled, 1749905, 1:300), rabbit anti HESX 1 (Abcam, AB246949, polyclonal, GR3267093-1, 1:100), goat anti-Brachyury (T)