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. 2022 Mar 7;54(3):318–327. doi: 10.1038/s41588-022-01023-0

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 4 — a. USP7 protein quantification upon Usp7 siRNA (corresponds to Fig.3a). Statistical comparisons: two-sided Welch’s t-test. b. Growth curve after Usp7 RNAi at day 1(D1) 2(D2) after seeding(D0). c. MERVL and Usp7 qRT-QPCR upon Usp7 RNAi. Statistical comparisons: two-sided Welch’s t-test. d. Venn diagram comparing downregulated genes in control, USP7-depleted ESCs, and USP7-depleted 2CLCs. e. logFC scatter plots between siUSP7-2CLCs and endogenous 2CLCs showing high overlap of upregulated genes (red) between both 2CLC samples. f. Gene-set enrichment analysis of siUSP7-induced 2CLCs against a ‘2C’ signature. g. PCA of siControl, siUSP7 ESCs (GFP⁻ cells) and siUSP7-induced 2CLCs transcriptomes compared with embryos and other 2CLC datasets. h. 2CLC percentage upon siRNA for Usp7 or HU treatment combined with siRNA for Dux. Statistical comparisons: two-sided Student’s t-test for pairwise comparison only between the indicated groups. i. Western Blot after Usp7 siRNA or control siRNA transfection and treatment of PLK1 inhibitor at indicated times. Statistical comparisons: two-sided Student’s t-test between each sample. Highest p-values are shown. j-k. Cell cycle profiles of ESCs (j) and 2CLC percentage (k) after transfection of control or Usp7 siRNA, followed by treatment with PLK1 inhibitor at indicated times. In (k) n for 42 h control samples is 2 biological replicates; for 48 h control is 4 and for all other samples is 5. Statistical comparisons between the indicated groups: two-sided Student’s t-test. l. Usp7 gene locus and knock-in strategy to insert Auxin-Inducible Degron (AID) at the C-terminus of USP7. m. Western Blot of USP7 after auxin (IAA) treatment. n. Western Blot in parental and knock-in USP7-AID line. The USP7-AID transgene causes lower USP7 expression compared to the parental clone, presumably leading to higher steady-state 2CLC population. o. Rrm1 and Rrm2 RT-qPCR 48 h after transfection with their respective siRNAs compared to control. Statistical comparisons: two-sided Welch’s t-test. p. Fork speed upon RNAi for RRM1, RRM2 or control. Statistical analysis: two-sided Wilcoxon rank-sum test. q. 2CLCs quantification 48 h after siRNA transfection. Statistical analysis: two-sided Student’s t-test. In bar graphs plots are mean±S.D and dots are individual replicate values. n: number of independent biological replicates. In m and n, dots are values from biological replicates.