Extended Data Fig. 6: Correction of chromatic aberrations and characterization of mESC lines with promoters flanking TetO and LacO arrays.
From: Cohesin and CTCF control the dynamics of chromosome folding
A. Bar plot showing the number of detected spots per cell per channel for 1,400 manually annotated images subsampled from the images series. In 3% of the images 2 spots per cell are detected indicating the presence of sister-chromatids. B. Distribution of pairwise distances in each dimension for co-localized signals measured on beads (n = 2,226 timepoints) or on the control TetO cell line (n = 69,453 timepoints), as well as for chromatic-aberration corrected and uncorrected images from TetO-LacO cell lines (in the presence of cohesin and 3xCTCF sites, n = 848,955 timepoints). Boxplot: boxes denote lower and upper quartiles (Q1 and Q3, respectively); whiskers denote 1.5x the interquartile region (IQR) below Q1 and above Q3. C. Schematic representation of the ‘Control TetO’ cell line that contains multiple TetO array integrations as well as stable integrations of TetR-eGFP and TetR-tdTomato. This allows labeling of each TetO array with two separate fluorophores. D. Representative images of the ‘Control TetO’ cell line. The time series shows a zoomed version of the region indicated by the white square. E. Radial distance distribution of the ‘Control TetO’ cell line as defined in panel C and D showing that the resolution on the 3D distance is ~130 nm. F. Schematic representation of cell line containing 3-phosphoglycerate kinase (PGK) promoters driving the expression of resistance gene directly adjacent to the operator arrays. The expression cassettes can be excised using Dre recombination or piggyBac transposition to yield the cell line with operator arrays only (PGK = PGK promoter, NeoR = Neomycin resistance gene, PuroR = Puromycin resistance gene, pA = polyadenylation signal, ITR = inverted terminal repeats for piggyBac recognition, Rox = Rox sites for Dre recombination). G. Differential map at 6.4 kb resolution for the structural differences between the E14 cell line containing LacO and TetO insertions with the adjacent promoters vs. the E14 cell line containing the operator arrays only (see Methods). Dashed lines indicate the insertion sites.
