REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
pERK p44/42 MAPK (T202/T204) XP	Cell Signaling Technology	Cat#4370S
Etv5	Abcam	Cat#ab102010; RRID:AB_10711030
Flag M2	Sigma-Aldrich	Cat#F1804; RRID:AB_262044
Rbpj	Cell Signaling Technology	Cat#5442S; RRID:AB_10695407
Normal rabbit IgG	Cell Signaling Technology	Cat#2729S
Normal rabbit IgG	Abcam	Cat#ab171870
Normal mouse IgG	Santa Cruz	Cat#sc-2025
GAPDH	Sigma-Aldrich	Cat#G8795; RRID:AB_1078991
H3K27Ac	Active Motif	Cat#39135; RRID:AB_2614979
ERK	Cell Signaling Technology	Cat#9107
Pou3f1 (Oct6) C-20	Santa Cruz	Cat#sc-11661; RRID: AB_2268536
Lef1	Abcam	Cat#ab137872
GATA4	Santa Cruz	Cat#sc1237; RRID: AB_2108747
Sox2	eBioscience	Cat#14-9811-80; RRID: AB_11219070
Tuj1	R&D Systems	Cat#MAB1195
Sox1	Cell Signaling Technology	Cat#4194; RRID:AB_1904140
Klf4	R& D Systems	Cat#AF3158; RRID:AB_2130245
Nanog	eBioscience	Cat#14-5761-80; RRID:AB_763613
Tfcp2l1	R& D Systems	Cat#AF572; RRID:AB_2202564
H3K4me3	Diagenode	Cat#pAb-MEHAHS-024
GFP	ThermoFisher Scientific	Cat#A-11122; RRID:AB_221569
T (Brachyury)	R&D Systems	Cat#AF2085; RRID:AB_2200235
Oct4	Santa Cruz	Cat#sc-5279; RRID:AB_628051
b-tubulin	Abcam	Cat#ab6046; RRID:AB_2210370
Chemicals, Peptides, and Recombinant Proteins
MEK inhibitor PD0325901	ABCR	Cat#AB 253775
GSK3 inhibitor CHIR99021	ABCR	Cat#AB 253776
Laminin	Millipore	Cat#CC095-5MG
Fibronectin	Millipore	Cat#FC010
ROCK inhibitor Y-27632	Merck Chemicals	Cat#688000-100MG
Tankyrase inhibitor XAV939	Cell Guidance Systems	Cat#SMS38-200
Activin A	Made in house	N/A
Fgf2	Made in house	N/A
LIF	Made in house	N/A
N2B27	Made in house	N/A
Accutase	Millipore	Cat#SCR005
Gelatin	Sigma-Aldrich	Cat#G1890
Γ-secretase inhibitor	Calbiochem	Cat#CAS 209984-56-5
Γ-secretase inhibitor DAPT	Merck Chemicals	Cat#565770
FuGENE HD transfection reagent	Promega	E2311
Critical Commercial Assays
Alkaline Phosphatase Kit	Sigma-Aldrich	Cat#86R-1KT
NEPER Nuclear Cytoplasm Extraction Reagents	ThermoFisher Scientific	Cat#78833
Dynabeads Protein G	Thermofisher Scientific	Cat#10004D
Deposited Data
Etv5 ChIP-seq (2i, N16h)	This study	GEO: GSE122338
H3K4me3 ChIP-seq (2i, N16h)	This study	GEO: GSE122338
RNA-seq of Etv5, Rbpj and Tcf3 single, double, and triple mutant ESCs	This study	GEO: GSE122338
Experimental Models: Cell Lines
Rex1-GFPd2 (RGd2) ESC	Kalkan et al., 2017	N/A
Rex1-GFPd2 c1 (RGd2-2) ESC (clonal line)	This study	N/A
Etv5 knockout ESC (3 clonal lines)	This study	N/A
Rbpj knockout ESC (3 clonal lines)	This study	N/A
Tcf3-knockout ESC (2 clonal lines)	This study	N/A
Etv5/Tcf3 double knockout ESC (2 clonal lines)	This study	N/A
Rbpj/Tcf3 double knockout ESC (2 clonal lines)	This study	N/A
Etv5/Tcf3/Rbpj triple knock out ESC (3 clonal lines)	This study	N/A
Etv4 knockout ESC (3 clonal lines)	This study	N/A
Etv4/Etv5 knockout lines (1 clonal line)	This study	N/A
Hes1 knockout ESC (2 clonal lines)	This study	N/A
Id3 knockout ESC (2 clonal lines)	This study	N/A
Lef1 knockout ESC (2 clonal lines)	This study	N/A
Pou3f1 knockout ESC (2 clonal lines)	This study	N/A
Pou3f1/Lef1 double knockout ESC (2 clonal lines)	This study	N/A
RGd2/mKusabira Orange (mKO) ESC	This study	N/A
RGd2/ H2B-tdTomato ESC	This study	N/A
ETR-tKO/mKusabira Orange(mKO) ESC	This study	N/A
ETR-tKO/H2B-tdTomato ESC	This study	N/A
Etv5-3xFlag knockin ESCs (2 clonal lines)	This study	N/A
Etv5-KO/Etv5-3xFLAG rescue ESC	This study	N/A
Etv5-KO/ΔEtv5-3xFLAG rescue ESC	This study	N/A
Etv5-KO/mKusabira Orange (mKO)	This study	N/A
Rbpj-KO/Rbpj rescue ESC	This study	N/A
ETR-tKO/TetG/TRE3G-iEpT ESC	This study	N/A
ETR-tKO/TetG ESC	This study	N/A
Etv4/Etv5 double knockout ESC (2 lines; PE15-3, PE15-4)	Lu et al., 2009	N/A
Etv4/Etv5 dKO (PE3/PE4)/ mKusabira Orange ESC	This study	N/A
Etv4/Etv5 dKO (PE3/PE4)/ Etv5-3xFlag rescue ESC	This study	N/A
Etv4/Etv5 dKO (PE3/PE4)/ ΔEtv5-3xFLAG rescue ESC	This study	N/A
Wild type ESC	Lu et al., 2009	N/A
Experimental Models: Organisms/Strains
Mus musculus females aged 6-10 weeks: CD1 strain was used to provide embryos and CBA/BL6 F1 animals were employed as transfer recipients for embryo chimeras.	N/A	N/A
Oligonucleotides
Nanog siRNA, Mm_Nanog_3 FlexiTube siRNA	QIAGEN	Cat#SI01323357
Nanog siRNA, Mm_LOC100038891_2 FlexiTube siRNA	QIAGEN	Cat#SI04460869
Tbx3 pre-designed siRNA Assay ID: 223884	Thermofisher Scientific	Cat#AM16708
Tbx3 pre-designed siRNA Assay ID 223885	Thermofisher Scientific	Cat#AM16708
gRNAs Sequences	See Table S7 for sequences	N/A
Taqman and UPL gene expression assays	See Table S7 for oligo sequences and catalog numbers	N/A
ChIP-qPCR Primer Sequences	See Table S7 for sequences	N/A
Recombinant DNA
TRE3G-Etv5-p2A-Tcf3-pGK-Hygo	This study	N/A
CAG-Etv5-3xFlag-pGK-Hygro	This study	N/A
CAG-ΔN-Etv5- 3xFlag-pGK-Hygro	This study	N/A
CAG-mKusabira Orange-pGK-Hygro	This study	N/A
CAG-Rbpj-pGK-Hygro	This study	N/A
CAG-H2B-tdTomato-IRES-Puromycin	This study	N/A
px459_SpCas9-2A-Puro	Addgene	#62988
Software and Algorithms
Bowtie2	Langmead and Salzberg, 2012	N/A
MACS2	Zhang et al., 2008	N/A
DeepTools	Ramírez et al., 2016	https://doi.org/10.1093/nar/gkw257
Trim Galore!		https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/
Bowtie2	Langmead and Salzberg, 2012	http://bowtie-bio.sourceforge.net/bowtie2/index.shtml
Samtools		http://samtools.sourceforge.net/
FeatureCounts	Liao et al., 2014	https://doi.org/10.1093/bioinformatics/btt656
R		https://www.R-project.org/
DESeq2		https://www.bioconductor.org/packages/release/bioc/html/DESeq2.html
Goseq		https://www.bioconductor.org/packages/release/bioc/html/goseq.html
Pheatmap		https://cran.r-project.org/web/packages/pheatmap/index.html