Extended Data Fig. 5. O-eLIT with JEB.

a) Chr19–9K. One round of O-LIT (SOLiD) or O-eLIT (JEB) off of Mainstreet. Maximum z-projections representative of 2 replicates.

b) Chr19–9K signal over nuclear background measurements after one round of O-LIT (orange; n = 113 puncta from 55 cells from 2 replicates) or O-eLIT (blue; n = 136 puncta from 57 cells from 2 replicates). Bar is the mean and SD.

c) Tier 1 detection of 36plex-1K after five rounds of O-LIT with SOLiD reagents (orange; average of 51.75%, n = 41) or O-eLIT with JEB (blue; average of 61.2 ± 10.2%, n = 440 from 9 replicates). Detection efficiency from individual replicates are plotted. Error bars represent 95% bootstrap confidence interval of the mean. 36plex-1K library shares first 1,000 Oligopaint oligos of each target in 36plex-5K. For example, for target 2pR1, 36plex-5K spans the chromosomal region from nt position 1,002,895 to 1,660,898 (~658 kb), whereas 36plex-1K spans the region from nt 1,002,895 to 1,147,495 (~144 kb).

d) FP discovery rate from panel c. SOLiD = 7.49% and JEB = 8.95%. FP discovery rate from individual replicates are plotted. Error bars represent 95% bootstrap confidence interval of the mean.

e) Chromosome traces and ball and stick of Fig. 4c cell after Tier 2 detection and five rounds of O-eLIT 36plex-1K. 63/66 (95%) targets were detected. Asterisks, beginning of chromosomes. n = 1.

f) Single-cell pairwise spatial distance matrices of panel C cell.

g) 36plex-1K population pairwise spatial distance measurements (top, from Fig. 3f). Average pairwise spatial distance from cell population after Tier 1 detection (n = 440 from 9 replicates). Measurements from homologous targets were combined. Bottom, Hi-C data of 36plex-5K targets obtained from (Nir et al. 2018).

h) 36plex-1K detection rate for individual cells from 9 replicates.

i) Percentage of cells displaying a range of efficiencies of barcode detection after 36plex-1K O-eLIT off of Mainstreet.