REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Human/Mouse Brachyury Affinity Purified Polyclonal Ab antibody	R and D Systems	Cat# AF2085; RRID: AB_2200235
Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647	Thermo Fisher Scientific	Cat# A-21447; RRID: AB_2535864
Chemicals, Peptides, and Recombinant Proteins
VECTASHIELD Mounting Medium	Vector Laboratories	Cat# H-1000; RRID: AB_2336789
Alexa Fluor 488 Phalloidin	Thermo Fisher Scientific	Cat# A12379
Critical Commercial Assays
Chromium Single Cell 3′ Library & Gel Bead Kit v2	10X Genomics	PN-120237
Chromium Single Cell 3′ Chip Kit v2	10X Genomics	PN-120236
Deposited Data
T chimera single-cell RNA-seq	This paper	ArrayExpress: E-MTAB-8811
New wild-type chimera single-cell RNA-seq	This paper	ArrayExpress: E-MTAB-8812
Existing wild-type chimera single-cell RNA-seq	Pijuan-Sala et al., 2019	ArrayExpress: E-MTAB-7324
Mouse gastrulation single-cell RNA-seq atlas	Pijuan-Sala et al., 2019	ArrayExpress: E-MTAB-6967
Experimental Models: Cell Lines
tdTomato+ mESC line (male, karyotypically normal)	Pijuan-Sala et al. 2019	B6xtdTom+ cl12-F10
T-/- tdTomato+ mESC Clone 1	This paper	B6xtdTom+ cl12-F10-B6
T-/- tdTomato+ mESC Clone 2	This paper	B6xtdTom+ cl12-F10-C6
Experimental Models: Organisms/Strains
Heterozygous T/+ BTBR/Pas mice	Rashbass et al., 1994	Heterozygous T/+ BTBR/Pas mice
C57BL/6 wild type mice	Charles River	C57BL/6J (JAX™ Mice Strain)
Oligonucleotides
F-primer for embryo genotyping (Figures S4I and S4J) CCAGTTGACACCGGTTGTTACA	Sigma Aldrich	N/A
R-primer for embryo genotyping (Figures S4I and S4J) TATCCCAGTCTCTGGTCTGT	Sigma Aldrich	N/A
F-primer for embryo genotyping (Figures S4I and S4J; positive control) GCGCCAGTGCAGGGAAGATTGGAA	Sigma Aldrich	N/A
R-primer for embryo genotyping (Figures S4I and S4J; positive control) GATATGACTGGGCCAGACGGAAA	Sigma Aldrich	N/A
T locus targeting gRNA1: TGACGGCTGACAACCACCGC	Sigma Aldrich	N/A
T locus targeting gRNA2: GCCCCAAAATTGGGCGAGTC	Sigma Aldrich	N/A
F-primer for NGS of T-targeted mESC clones TCGTCGGCAGCGTCAGATGTGTATAA GAGACAGTCCCGGTGCTGAAGGTAAAT	Sigma Aldrich	N/A
R-primer for NGS of T-targeted mESC clones GTCTCGTGGGCTCGGAGATGTGTA TAAGAGACAGCCTGCTTAACCC TCATCAGC	Sigma Aldrich	N/A
Recombinant DNA
pX458 plasmid	Addgene	#48138
Software and Algorithms
Cellranger	Zheng et al., 2017	https://support.10xgenomics.com/single-cell-gene-expression/software/downloads/latest?
Scran	Lun et al., 2016	http://bioconductor.org/packages/release/bioc/html/scran.html
DropletUtils	Griffiths et al., 2018	http://bioconductor.org/packages/release/bioc/html/DropletUtils.html
Uwot	McInnes et al. 2020, https://arxiv.org/abs/1802.03426	https://cran.r-project.org/web/packages/uwot/index.html
Destiny	Angerer et al. 2016	http://bioconductor.org/packages/release/bioc/html/destiny.html
FIJI	Schindelin et al. 2012	https://imagej.net/Fiji