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. 2022 Jul 21;54(8):1238–1247. doi: 10.1038/s41588-022-01132-w

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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. 2 — a. Analyses of cells used in Fig. 1j. (left) RNAPII IF intensity at the miR290-295 FISH foci (n_DMSO = 61, n_dTAG-13 = 50). (middle) RNAPII mean fluorescence intensity at random nuclear positions (n_DMSO = 61, n_dTAG-13 = 50). (right) Distance between the FISH focus and the nearest RNAPII puncta (n_DMSO = 67, n_dTAG-13 = 53). Data presented as mean values ± SD from one staining experiment. P values are from two-sided Mann-Whitney tests. NS: not significant. b. Images of RNA-FISH and IF signal. Nuclear periphery determined by DAPI staining is highlighted as a white contour. Also shown are averaged signal of either RNA FISH or RNAPII IF centered on the miR290-295 FISH foci or randomly selected nuclear positions. Data were collected as an independent replicate of experiments displayed in Fig. 1j. Scale bars: 2.5 μm. c. Analysis of cells used in panel ‘b’. (left) RNAPII IF intensity at the miR290-295 FISH foci (n_DMSO = 43, n_dTAG-13 = 25). (center) RNAPII mean fluorescence intensity (n_DMSO = 30, n_dTAG-13 = 40). (right) Number of RNAPII puncta on a representative set of cells (n_DMSO = 22, n_dTAG-13 = 22). Data are presented as mean values ± SD from one staining experiment. P values are from two-sided Mann-Whitney tests. NS: not significant. d. Images of individual z-slices (same z) of the Fgf4 RNA-FISH and IF signal. Nuclear periphery determined by DAPI staining is highlighted as a white contour. Also shown are averaged signals of either RNA FISH or RNAPII IF centered on the FISH foci or randomly selected positions. Scale bars: 2.5 μm. e. Analyses of cells used in panel ‘d.’ (left) RNAPII IF intensity at the Fgf4 FISH foci (n_DMSO = 53, n_dTAG-13 = 37). (right) RNAPII mean fluorescence intensity at random nuclear positions (n_DMSO = 53, n_dTAG-13 = 29). Data presented as mean values ± SD from one staining experiment. P values are from two-sided Mann-Whitney tests. NS: not significant. f. (left) Scheme of FKBP knock-in strategy in the R1 mESCs used in the PALM experiments. (right) TRIM28 Western blot in the R1 mESCs. Western blot was done once.

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