3'-deoxy-5-(cyanine dye 3)uridine 5'-trisphosphate

For the self versus self hybridizations, custom cDNA microarray experiments proceed as follows. Altogether, three microarray hybridizations were performed using custom printed cDNA microarray slides from the same print batch. Labelling, hybridization and washing were done as described previously by Monni et al. [40 (link)] and Järvinen et al. [20 (link)]. Briefly, total RNA was extracted from cell lines BT-474, HBL-100, and MCF-7 and labelled with Cy3-dUTP and Cy5-dUTP (Amersham Biosciences, Piscataway, NJ). The custom printed cDNA microarrays comprised of 11,520 clones from Incyte Genomics IRAL cDNA library and 1,136 clones from Research Genetics library. Microarrays were printed on poly-l-lysine coated slides using an Omnigrid arrayer (GeneMachines) as described previously [20 (link)]. Microarrays were scanned with an Agilent laser confocal scanner (Agilent Technologies, Palo Alto, CA) and gridded using the DEARRAY software developed by Chen et al. [29 (link)]. For the four breast cancer cell lines, custom cDNA microarray experiments were provided in a separate contribution by Järvinen et al. [20 (link)] and detailed protocols are described in that work. The relevant genes in our study were verified using RT-PCR in a parallel study by Hyman et al. [35 (link)].

For metaphase spread preparation, mitoses were mechanically collected by blowing the medium on the dish surface. Chromosome preparations were performed with the standard air-drying procedure and were used for karyotype analysis or fluorescence in situ hybridization (FISH).
Whole genomic DNA from Przewalski’s horse fibroblasts was extracted according to standard procedures. BAC clones (CHOR1241-17G8, coordinates in EquCab2.0: chr5:37,011,456–37,167,308; CHOR1241-25H7 coordinates in EquCab2.0: chr5: 98,031,303–98,224,666) and lambda phage 37cen and 2PI clones [16 (link),53 (link)] were extracted from 10 mL bacterial cultures with the Quantum Prep Plasmid miniprep kit (BioRad, Milan, Italy), according to supplier instructions. All probes were labeled by nick translation with Cy3-dUTP (Enzo Life Sciences, Milan, Italy) or Alexa488-dUTP (Life Technologies, Monza, Italy) as previously described [64 (link)]. FISH was performed as previously described [68 (link)].
Chromosomes were counterstained by DAPI. Digital grayscale images for fluorescence signals were acquired with a fluorescence microscope (Zeiss Axio Scope.A1, Zeiss, Göttingen, Germany) equipped with a cooled CCD camera (Teledyne Photometrics, Birmingham, UK). Pseudocoloring and merging of images were performed using the IPLab 3.5.5 Imaging Software (Scanalytics Inc., Fairfax, VA, USA). Chromosomes were identified by DAPI banding according to the published karyotypes [49 (link),50 (link)].

Primers for the six most abundant sequences of transposable elements (LTR retrotransposons Ty1/copia SIRE, Tork and TAR, and LTR retrotransposons Ty3/gypsy Ogre, Athila and Chromovirus) were designed based on the Integrase domain using the Geneious 7.0 software (Supplementary Table S1). For the satellite DNA family, primers were designed facing outwards, anchored at the most conserved region of the monomer, using the same software. The repetitive sequences were amplified by PCR under the following conditions: initial denaturation at 94 °C for 3 min, followed by 30 amplification cycles, each consisting of 94 °C for 1 min, 60 °C for 1 min and 72 °C for 1 min, and 10 min final elongation at 72 °C. PCR reactions included 50 ng genomic DNA, 0.5 μM of each primer, 0.1 mM dNTP, 1× PCR buffer (Invitrogen, Waltham, MA, USA), 2 mM MgCl₂, 1× TBT (50 mM trehalose, 1 mg/mL BSA, 1% Tween 20 and 8.5 mM Tris hydrochloride) and 0.04 U of Taq Polymerase (Invitrogen). Products were checked on a 1% agarose gel, and sequences were confirmed and labelled with Cy3-dUTP (GE) by nick translation (Roche, Basel, Switzerland).
To locate the ribosomal DNA sites, a clone containing a 500 bp fragment of the 5S rDNA of Lotus japonicus (Regel) K.Larsen (Fabaceae) [41 (link)] was labelled with Cy3-dUTP (GE), and a clone containing a 6.5 kb fragment corresponding to the 35S rDNA of Arabidopsis thaliana [42 (link)] was labelled with digoxigenin-11-dUTP (Roche). The 35S rDNA probe was detected with anti-digoxigenin produced in sheep, conjugated with FITC (Roche), and signals were amplified with anti-sheep IgG produced in rabbits also conjugated with FITC (Serotec).
FISH was performed according to [41 (link)], with minor modifications. The hybridization mix was composed of 50% formamide (v/v), 10% dextran sulphate (w/v), 2× SSC and 50 ng/μL of each probe. The slides were denatured at 75 °C for 3 min, and the final stringency of hybridization was 76%.

Conspicuous aberrant regions with the most frequent changes in the examined ECs were defined as target regions. Within these target regions, we have identified potential bacterial artificial chromosomes (BACs) that could be considered as possible FISH samples. We obtained corresponding BAC clones from BACPAC Genomics Inc (Richmond, Ca) in lysogeny broth (LB) agar stab cultures. These DH10B Escherichia coli clones were grown overnight in LB with 12.5 μg/ml chloramphenicol at 37°C and periodically shaken. BAC DNA was isolated with the Qiagen® Plasmid Mini Kit (Hilden) following the manufacturer's protocol. As described by Weimer et al., the BAC DNA was amplified and labelled with fluorescent dyes by DOP‐PCR.¹⁶ RP11‐349C9, RP11‐474P6, RP11‐186B18 and RP11‐178D22 were labelled with dUTP‐Cy3. RP11‐962I23, RP11‐124 K18, RP11‐60P20 and RP11‐472P14 were labelled with green‐dUTP. RP11‐90 L5, RP11‐54D8 and RP11‐61H16 were labelled with green‐dUTP and Cy3‐dUTP. The labelled PCR products were combined and supplemented with 40 μg Cot‐1 DNA (GeneOn) and precipitated in 70% ethanol overnight. The precipitate was centrifuged for 30 min at 13,000 RPI, the supernatant was discarded and the pellet was freeze‐dried. The dry pellet was dissolved in 50 μl hybridization solution (2× SSC, 50% formamide, 10% Dextran sulphate, 1% Tween 20, pH 7.0).