Fluorescence in situ hybridization (FISH)

FISH was performed as previously described¹⁰. Briefly, LTED cells grown on coverslips were fixed with 4% paraformaldehyde and 0.5% Triton X-100 in PBS for 15 min at room temperature and then permeabilized with 0.5% saponin and 0.5% Triton X-100 in PBS for 20 min at room temperature. The coverslips were immersed in 20% glycerol in PBS for 30 min at room temperature and then subjected to four cycles of freezing and thawing. In a cycle, the coverslips were frozen in liquid nitrogen for 3 s followed by being thawed at room temperature. The cells were placed back in 20% glycerol and then treated with 0.1 N HCl for 15 min at room temperature. For denaturation and hybridization, the cells were incubated in hybridization mixtures (2 × SSC, 50% formamide, 10% dextran sulfate, 1 mg/mL tRNA, and 5–10 μg/mL probe DNA) at 37 °C for over 48 h with moisture in a Hybridizer (Dako). The BAC probes (RP11–450E24 for Figs 2, ​,4,4, ​,5,5, Supplementary Figs ^S1 and ^S3; RP1-63I5 for Supplementary Fig. ^S5) were labeled with digoxigenin (for Figs 2, ​,44 and ​and5)5) or biotin (for Supplementary Fig. ^S5) in a nick translation mixture (Roche) according to the manufacturer’s protocol. After hybridization, the coverslips were washed three times with 2 × SSC and 50% formamide at 37 °C for 5 min at room temperature, followed by another three washes with 2 × SSC at 37 °C for 5 min. The cells were further incubated with FITC-anti-digoxigenin (Roche, for Figs 2, ​,4,4, ​,55 and Supplementary Figs ^S1 and ^S3) or Alexa Fluor 488 Streptavidine (Invitrogen Molecular Probes, for Supplementary Fig. ^S5) to detect signals. DNA was counterstained with 5,6-diamidino-2-phenylindole (DAPI). Lastly, the coverslips were mounted on a slide with ProLong Gold Antifade (Thermo Fisher Scientific) for observation under a microscope.