A probe for Xist RNA was prepared by using Nick translation reagent kit (Abbott Molecular, 07J00-001) with Cy3-dCTP (GE Healthcare, PA53021). The template DNA was a plasmid coding the full-length mouse Xist gene (Addgene, 26760) (Wutz and Jaenisch 2000 (link)). A probe for DNA FISH was prepared using the same kit with Green-dUTP (Abbott Molecular, 02N32-050). The template DNA was a BAC clone containing the Rnf12 locus (RP23-36C20) (Fukuda et al. 2015 (link)). The fluorescent probes were ethanol-precipitated with 5 µg of Cot-1 DNA (Life technologies), 5 µg of herring sperm DNA (Thermo Fisher Scientific), and 2.5 µg of yeast tRNA (Thermo Fisher Scientific, AM7119) and then dissolved with 20 µL of formamide (Thermo Fisher Scientific, 17899). The probes were stored at 4°C. Before being used, the probes (0.75 µL each) were mixed with 0.75 µL of Cot-1 DNA/formamide and 2.25 µL of 4× SSC/20% dextran (Millipore S4030). The probe mixtures were heated for 30 min at 80°C and then transferred to a 37°C incubator (“preannealed probes”).