Extended Data Figure 1. Effect of auxin treatment on mESCs.

(a) Schematic of Shh and Lmbr1 genes showing the position of directly labelled Custom Stellaris® RNA FISH oligo probes used for RNA FISH. Shh probes were labelled with Quasar 670 and Lmbr1 probe with Quasar 570 (b) Images of representative nuclei showing RNA FISH signals for Shh (white) and Lmbr1 (red) probes from wild type mESCs transfected with tShh-VP64 or tShh-Δ, and either untreated (- auxin) or treated with 24 hours of auxin (+ auxin). Shh RNA FISH signal is indicated by white arrow. Scale bars = 5 μm. (c) Quantification of the percent of (left) Shh (pink and red bars) and (right) Lmbr1 – intron 1 (white and grey bars) expressing alleles in mESCs transfected with tShh-VP64, tSBE2-VP64 and tZRS-VP64 and equivalent TALE-Δ controls. Cells were either untreated (- auxin) or treated with 24 hours of auxin (+ auxin). Biological replicate of the data shown in Fig. 1f. The data were compared using a two-sided Fisher’s Exact Test, n = numer of alleles scored, ns – not significant. (d) Table showing two-sided Fisher’s Exact Test p-values for differences in the percent of Shh-expressing alleles in mESCs transfected with TALE-Vp64 or TALE-Δ constructs assayed by RNA FISH in the absence or presence of auxin. Data from Figure 1f and Extended Data Figure 1c. p-values in bold are significant (<0.05). (e) Quantification of live cells by DAPI staining during flow cytometry in untreated and auxin-treated wild type (WT), CTCF-AID and SCC1-AID cells after 6, 24 and 48 hours of growth in auxin. Source Data Extended Data Fig1.