Extended Data Fig. 9: Generation of Stag3-only ESCs.

a, FACS selection of ESCs expressing eGFP and mCherry2 10 days after transfection of plasmids expressing Cas9 and sgRNA targeting the Rosa26 locus and the knock-in cassette as shown in Fig. 6a. b, (left) Scheme for the detection of the knock-in allele by four PCR pairs. (right) PCR bands of each primer pair on agarose gel electrophoresis. c, Efficiency of the knockouts by frame-shift mutations using either Stag1- or Stag2-sgRNAs in WT ESCs. Eight clones were assessed for each KO, and the efficiencies for any-base insertion or deletion were calculated. In-frame deletions were regarded as WT. d, (left) Scheme for the generation of Stag1/2 DKO ESCs on Stag1 KO- Stag3 over-expressing ESCs with Stag2 sgRNA. (right) KO efficiency with frame-shift mutations. Thirty-two clones were assessed, and the efficiency with any-base insertion or deletion in the Stag2 allele was calculated. In-frame deletions were regarded as WT. e, Western blot for STAG1, STAG2, eGFP-STAG3, and α-tubulin in WT GSCs and Stag3-only ESCs using the whole cell lysate (one experiment). f, Enrichment of the homotypic (that is, intra-compartmental) interaction strength measured by cooltools¹⁵² in WT and Stag3-only ESCs. g, Correlation of differential insulation scores between WT and Stag3-only ESCs with those between CTCF, WAPL, or RAD21 non-degraded and degraded ESCs^47,65,66,67. Treated: auxin-treated for AID-tagged RAD21, CTCF, or WAPL. AID: auxin-inducible degron. h, Representative Western blot image for RAD21 and histone H3 expression in WT and Stag3-only ESCs using the chromatin fraction. Two biological replicates in each sample were analyzed. i, Abundance of RAD21 as quantified in (h) normalized by histone H3. j, Western blot for RAD21 and histone H3 expression in Stag1-only and Stag3-only mRAD21-Halo ESCs using the chromatin fraction (one experiment). While Stag1-only ESCs retain the intact Stag3 gene, they showed negligible STAG3 expression.

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