Cy5 dctp

Array CGH analysis was performed using standard methods described [5 (link)]. In brief, 300 ng of genomic DNA was labeled with Cy3-dCTP or Cy5-dCTP (GE Healthcare, Belgium) using Bioprime array CGH genomic labeling system (Invitrogen, Belgium). For the labeling, we used the “triangle method”: DNA samples from patients and controls were labeled and hybridized using a dye swap in trios consisting of at least one control per triangle. Samples were hybridized on 244K arrays (design ID 014693, Agilent, Belgium) for 40 h at 65°C. After washing, the samples were scanned at 5 μm resolution using a DNA microarray scanner G2505B (Agilent, Belgium). The scan images were analyzed using the feature extraction software 9.5.3.1 (Agilent) and further analyzed with “arrayCGHbase” [6 (link)]. Copy number variations were taken into consideration when two or more flanking probes were exceeding a value of the intensity ratios ± four times the standard deviation of log₂ of all intensity ratios for that experiment. Always two experiments investigating the same sample with a dye swap were compared and only when an alteration is present in both experiments was the region included for further analysis. Inconsistencies were inspected manually.

Oligonucleotides used for EMSA are listed in Supplementary Table 6. For AP1, AP1m1, AP1m2, AGI-II and S-AGI, complementary single-stranded oligonucleotides were annealed in annealing buffer (10 mM Tris pH 7.5, 150 mM NaCl and 1 mM EDTA). The resulting double-stranded DNA with a protruding G was fluorescently labelled by end filling: 4 pmol of double-stranded DNA was incubated with 1 unit of Klenow fragment polymerase (Ozyme) and 8 pmol Cy3-dCTP or Cy5-dCTP (GE Healthcare) in Klenow buffer during 2 h at 37 °C, followed by 10 min enzyme inactivation at 65 °C. Binding reactions were performed in 20 μl binding buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% glycerol, 0.25 mM EDTA, 2 mM MgCl₂, 0,01% Tween-20 and 3 mM TCEP) with 10 nM labelled probe, 1 × (28 ng ml⁻¹) fish sperm DNA (Roche) as nonspecific competitor and 25–500 nM proteins.
Competition assays were performed in duplicates and 1–100 × fish sperm DNA (Roche) was used in the binding reaction. Signal quantification was performed in using ImageLab v2.0.1 (Bio-Rad Laboratories). Signal of each protein–DNA complex was quantified relatively to total DNA signal. For Fig. 4e, each binding reaction was performed in triplicate. Uncropped gels are presented as Supplementary Fig. 10.

Human and mouse probes [XIST BAC (BAC PAC, RP13-183A17); ATRX BAC (BAC PAC, RP11-42M11); USP9X BAC (Invitrogen, CTD 3174G14); XIST fosmid (BAC PAC, G135P63425C4)] were labeled with Fluorescein-12-dUTP (Invitrogen), Cy3-dCTP (GE Healthcare, #PA53021), or Cy5-dCTP (GE Healthcare, #PA55031). Labeled probes for multiple genes were precipitated in a 3 M sodium acetate (Teknova, #S0298) solution along with 300 μg of yeast tRNA (Invitrogen, #15401–029), and 150 μg of sheared, boiled salmon sperm DNA (Invitrogen, #15632–011). The solution was then centrifuged at 21,130 X g for 20 min at 4 °C. The resulting pellet was washed in 70% ethanol, then washed in 100% ethanol, dried, and re-suspended in deionized formamide (ISC Bioexpress, #0606–500 ML). The re-suspended probe was denatured via incubation at 90 °C for 10 min followed by an immediate 5 min incubation on ice. A 2X hybridization solution consisting of 4X SSC, 20% Dextran sulfate (Millipore, #S4030), and 2.5 mg/ml purified BSA (New England Biolabs, #B9001S) was added to the denatured probe/formamide solution. Probes were stored at −20 °C until use.