Extended Data Fig. 9. O-eLIT comparison of X chromosomes in female IMR-90 cells after ChrX-46plex-2K O-eLIT off of both streets.

a) First round of O-eLIT sequencing. MacroH2A.1 immunostaining after five rounds of O-eLIT marks the Xi. n = 1.

b-c) Xi and Xa traces (b) and ball and stick (c) of panel a nucleus after Tier 2 analysis and interpolation of missing targets. Sphere color corresponds to chromosome cartoon. n = 1.

d) Single-cell pairwise spatial distances after interpolation of missing targets in panel a.

e) Tier 2 target detection efficiency after five rounds of O-eLIT. 38.57% of targets are detected in 71 cells. Detection efficiency from individual replicates are plotted. Error bars represent 95% bootstrap confidence interval of the mean.

f) Population pairwise spatial distances after Tier 1 detection (n = 71 cells) and Hi-C data of IMR-90 cells (Rao et al. 2014).

g) Population contact maps (top) where two targets are considered to be in contact if less than 2 μm apart (n = 315 chromosomes). Bottom, Hi-C data as in panel f. (Spearman’s rank correlation with the Hi-C matrix is r =0.733, two-sided p-value for a hypothesis test whose null hypothesis is that two sets of data are uncorrelated = 2.564 × 10e-175).

h) Radius of gyration for the Xi (n = 40 chromosomes) and Xa (n = 31 chromosomes). The thick line in each violin plot represents the Interquartile range (IQR), the white dot marks the median and the thin lines extend 1.5 times the IQR. P-value = 7.08 × 10e-6 (two-sided t-test whose null hypothesis is equal means).

i-j) Linear plot of the mean spatial distance versus the genomic distance for all pairwise targets for Xi (n = 40 chromosomes) and Xa (n = 31 chromosomes).

k-l) Population contact maps for Xi (n = 40 chromosomes) and Xa (n = 31 chromosomes) with eigenvector analysis used to identify different domains. X1-X18 (white) and X19-X46 (grey) targets p and q arms, respectively.