Supplementary Materials and Methods Immunofluorescence, RNA FISH and DNA FISH on mouse embryos and testis cells The preparation of pre-implantation embryos, immunofluorescence and RNA FISH were carried out as described previously6. Probes used for RNA FISH were overlapping lambda clones for Chic121; a genomic BAC probe (RP24-148H21) for Smcx; and a cDNA probe for Ube1x (a kind gift from M. Sugimoto and N. Takagi). To identify the transgenic locus or a chromosome, DNA FISH was performed following immunofluorescence and/or RNA FISH. Images of embryos were first acquired, immediately following IF / RNA FISH. The coverslips carrying the embryos were then recovered in dH2O and incubated in Rnase A (100 ug/ml) in 2x SSC at 37 °C for 1h, before a brief rinse in 2x SSC, dehydration through an ethanol series (70%, 90%, 100%) and air drying. The DNA was then denatured in 70% formamide, 2x SSC for 10 min at 80 °C and dehydrated again through an ice-cold ethanol series. Hybridization with an Xic probe (YAC PA2) or a transgene specific YAC vector probe (pYAC) has been described previously18. Hybridization and revelation of the chromosome paint probes were performed according to manufacturer’s instructions (Cambio). Cover slips were counterstained with DAPI (1ug/ml), mounted and viewed under the fluorescence microscope. Mouse testes were isolated from 6-7-week –old male transgenic mice. Seminiferous tubules were minced into small fragments with small scissors in PBS to release spermatogenic cells, which were then transferred onto a glass coverslip in a drop of PBS and allowed to adhere by aspirating the excess medium prior to fixation. Specimen preparation for immunofluorescence or RNA FISH was carried out as described above. Primary antibody incubation was overnight at 4 °C, followed by a secondary antibody incubation of 30 min at RT. After washing in PBS, preparations were post-fixed in 4% paraformaldehyde for 10 min at RT and rinsed in PBS. Preparations were incubated in 0.7% Triton X-100, 0.1M HCl for 10 min on ice. They were then washed twice in 2x SSC for 10 min at RT. DNA FISH with paint probes for chromosomes 13 and X was carried out as described above, expect that the denaturation step was for 30 min at 80 °C; for pYAC4 and YAC PA-2 probes denaturation was for 5 min at 75°C. Preparation of metaphase spreads from ear fibroblasts and DNA FISH Ear fibroblasts were collected from ear tip tissue derived from male mice that were produced by setting up Tg 53 hemizygous intercrosses. Pieces of ear tip tissue were attached to the bottom of plastic tissue culture dishes and left to dry at room temperature. About 30 min later, DMEM with 10 % FBS was added. The ear tip tissues was cultured in a humidified atomosphere of 5% CO2 /95% air at 37 °C for 7-10 days. After 2 to 3 passages, metaphase chromosome preparation and DNA FISH were performed as described previously (S1, 18), with a denaturation step of 5 min at 75 °C 3D Microscopy Confocal image series were recorded on a LEICA TCS SP2 laser scanning microscope using the wavelengths 364 nm (Ar), 488 nm (Ar) and 543 nm (HeNe). Widefield 3D stacks were performed on a Delta Vision (Applied Precision Inc.) and deconvolved using Delta Vision sofware (Ratio method, 7 iterations). All optical sections were acquired by using a 100x objective and separate by 0.2 µm. Replication Timing analysis For late replication timing assays, embryos (late blastocysts) were placed in Eagle’s minimum essential medium (MEM) with 10% fetal bovine serum(FBS) containing 5-bromo-2’-deoxyuridine (BrdU, 7.5 ug/ml) and incubated for 6 h. For early replication timing assays S2, the late blastocysts were incubated in the presence of BrdU (7.5 ug/ml) for 1h and then transfered to MEM + 10% FBS with no BrdU, and incubated for another 9 hr. Incubation times were established in preliminary experiments, and were carried out based on previously published blastocyst cell cycle timesS3. Colcemid (0.03 ug/ml) was added to the medium for the last 1.5-2.0 h of incubation in all cases. Chromosome slides were prepared as described previouslyS3. Chromosome preparations were treated with 0.01% pepsin in 0.01N HCl at 37 °C for 7.5 min and then dehydrated in an ethanol series (70, 90 and 100%). Denaturation was then performed in 70% formamide/2xSSC for 2 min at 70°C followed by dehydration in an ice-cold ethanol series. After air-drying for 1h, the slides were incubated with blocking solution ( 1% BSA, 0.1% Tween 20 /PBS) for 10 min and then with mouse monoclonal anti-BrdU antibody (Becton Dickinson), diluted 1:200 in blocking solution, at 37°C for 30 min. The slides were then washed in PBST (0.1% Tween 20, PBS) three times for 5 min each and incubated for 30 min with Alexa Fluor 488 goat anti mouse IgG (Molecular Probes) appropriately diluted with blocking solution. After three further washes with PBST, the slides were mounted and the coverslips temporarily sealed. Slides were examined under the fluorescence microscope and selected images were captured. Coverslips were then removed in dH2O and the slides were washed in PBST, dehydrated in ethanol series, dried, denatured in 70% formamide/2xSSC for 2 min at 80°C and dehydrated in an ice-cold ethanol series, and subjected to chromosome painting with paint probes for Ch 13 or X (Cambio). Slides were stained with DAPI, mounted and viewed under the fluorescence microscope. Supplementary References S1. Popescu, P., Hayes, H. & Dutrillaux, B (Eds.). Techniques in animal cytogenetics. SpringerVerg. (1998). S2. Sugawara O., Takagi N. & Sasaki M. Allocyclic early replicating X chromosome in mice: genetic inactivity and shift into a late replicator in early embrogenesis. Chromosoma. 88,133138 (1983). S3. Dyban, A.P. An improved method for chromosome preparations from preimplantation mammalian embryos, oocytes or isolated blastocysts. Stain Technol. 58, 69-728 (1983). S4. Mukherjee, Anil.B. Cell cycle analysis and X-chromosome inactivation in the developing mouse. Proc Natl Acad Sci USA. 73, 1608-1611 (1976).