Testing whether FRET-FISH probes disrupt the target locus conformation

We co-hybridized each of the six different FRET-FISH probes designed to target the mouse Ogt gene (I1S50, I1S150, I12S50, I2S300, I4S50, I4S300) with two probes flanking the Ogt gene in female MEFs (see Supplementary Data ¹ for the list of oligos in each probe and Supplementary Fig. ^2a for a scheme of the oligo distribution and span of different Ogt probe designs). For the Ogt probes, we hybridized only the primary oligos in each probe, i.e., we did not add their detection oligos to the second hybridization mix, in which we only included fluorescently labeled oligos detecting the flanking probes. As control, we hybridized the two probes flanking the Ogt locus without any FRET-FISH probe. We imaged all the samples together with fluorescent beads sample, which we then used to correct the shift on the images. We used our in-house image analysis suite DOTTER to detect FISH dots and assign them to individually segmented nuclei. We used a custom script to calculate the Euclidean distance (in 3D) between any two dots corresponding to the two Ogt flanking probes using the following equation: