Fig. 2: Comparison of denaturing and non-denaturing FISH.

a Schematic of denaturing vs. non-denaturing FISH. b Two representative individual traces of 300 kb regions on chr 17 traced at 10 kb resolution with each FISH method in RPE-1 cells. Data representative of n = 1271 and n = 1514 cells from two independent experiments. Scale bar 100 nm. c Median pairwise distance maps of the same region as in b with denaturing (left column, n = 463 traces) and non-denaturing (middle column n = 503 traces). HiC contact frequencies (right column) from publicly available RPE-1 HiC data (GSE71831). Only cells assigned to G1 phase by DAPI intensity were used (~35%). d Spatial distance scaling of the same region as in (c) with the two FISH approaches. Shaded areas indicate 95 % confidence intervals around the median line. e Representative pairwise distance distributions between two consecutive 10 kb positions for the two FISH approaches. The histogram is overlayed by a kernel density estimate. Number of G1 traces analysed in (c−e) as indicated in (c). f Standard deviation of all pairwise distances from 300 kb regions on four chromosomes with the two FISH approaches. Boxes indicate the lower and upper quartiles and whisker indicate the data range with 1.5 times interquartile range (IQR). Number of traces per condition as indicated in Supplementary Fig. S2a. g Distribution of trace-completeness measured by percentage of spots passing quality control thresholds for denaturing (n = 2575 traces), and non-denaturing (n = 4505 traces) conditions after removal of systematically under-performing barcodes. The histogram is overlayed by a kernel density estimate. h The integrated DAPI intensity in the central z-plane per nuclei was used to assign cells to G1 or G2. i Correlation between median pairwise distances in a 300 kb region from cells separated into G1 and G2 populations with the non-denaturing FISH approach. Linear regression (blue line) and the resulting slope (s) and Pearson’s r are indicated. Data in (b−i) is representative of two independent experiments per condition. Source data are provided as a Source Data file.