Cy3 dutp

Unique 60-mer oligonucleotide probes were designed for each of the >56,000 high quality unigenes identified in this work. 8×60-K DNA microarray chips were implemented with the Agilent platform. A total of 12 sample RNAs consisting of one of each of the three different P levels from each experimental duration were further purified using an RNA Cleanup Kit (Macherey-Nagel, Germany). Total RNA was used with a cDNA Amplification Tag Kit (Jingxin, CapitalBio Corporation, China) and a NucleoSpinExtract II Kit (MN) to generate the targets. Each mRNA sample was reverse-transcribed in the presence of Cy3-dUTP or Cy5-dUTP (GE Healthcare) and 5 µg of labeled cDNA was hybridized with the microarray. After washing, microarray plates were dried briefly and scanned with an Agilent G2565CA Microarray Scanner. The ratios of the two fluorescent signal intensities of each DNA element were then measured to determine changes in gene expression. Microarray data were processed at the Biochip National Engineering Research Center of Beijing, CapitalBio Corporation. Analysis was performed using Feature Extraction and GeneSpring GX software with RMA normalization. Genes were considered differentially expressed if up-regulated by >2-fold or down-regulated by <0.5-fold (P<0.05). Microarray data have been deposited in the Gene Expression Omnibus database (accession number GSE52835).

The zona pellucida of embryos was removed using acid Tyrode solution (Sigma-Aldrich) and then fixed and permeabilized with 2% PFA-PVA containing 0.25% Triton X-100 for 10 min on ice. The samples were placed on glass slides, evaporated to dryness, dehydrated sequentially in 70 and 100% ethanol and then air-dried. Hybridization buffer containing an Xist probe (provided by T. Sado) was prepared using a Nick Translation Kit (Abbott, Abbott Park, IL, USA) and Cy3-dUTP (GE Healthcare Life Sciences, Fairfield, CT, USA) and was then applied to the slides. The slides were then incubated and washed as previously described26 (link). Fluorescence was visualized using the LSM510.

Spreading of mitotic chromosomes was carried out according to Hoang^{29 (link)}. 5S rDNA, 18S and 26S rDNA probes were generated from S. polyrhiza and from S. intermedia genomic DNA each by using designed primer pairs^{16 (link),21 (link),65 (link),66 (link)} as described^{24 (link)}. Ribosomal DNA, A. thaliana type telomere and S. polyrhiza BAC probes were labeled with Cy3-dUTP (GE Healthcare Life Science), Alexa Fluor 488-5-dUTP, Texas red-12-dUTP, biotin-dUTP or digoxigenin-dUTP (Life Technologies) and precipitated as described^{29 (link)}.
Denaturation of mitotic chromosomes and probes, hybridization, post-hybridization washing and signal detection were carried out according to Lysak et al.^{67 (link)}. Probe stripping and re-hybridization were done as described^{29 (link)}.

A Random Primer labeling Kit (RPLK): (Invitrogen, catalog number: 18187–013) was used to label each probe with Cy3-dUTP (GE Healthcare, catalog number: PA53022) for the 5′-flank of a supercontig probe, or with Cy5-dUTP (GE Healthcare, catalog number: PA55022) for the 3′-flank of a supercontig probe (GE Healthcare PA53022 and PA55022 respectively). From 500 to 1000 ng of probe DNA was labeled as proposed by the RPLK protocol. Each probe was resuspended in 10 μl of 2x Hybridization buffer, with the buffer recipe based on that described in [21 ]. Two probes of distinct colors, each corresponding to one of the supercontig’s flanks, were mixed and hybridized simultaneously to polytene chromosomes. To simplify mapping, chromosome images were taken before and after FISH.

Genomic DNA of each cell line was analyzed using Array CGH with the latest version of OncoBAC arrays for DNA copy. The OncoBAC arrays were comprised of 2402 P1, PAC, or BAC clones^{39 (link)}. About 80% of the clones on the OncoBAC arrays contained genes and STSs implicated in cancer development or progression. All clones were printed in quadruplicate. DNA samples for array CGH were labeled generally as described previously^{40 (link)–42 (link)}. Briefly, we random-prime labeled 500 ng of test (cell line) and reference (normal female, Promega) genomic DNA with CY3-dUTP and CY5-dUTP (GE Healthcare Life Science), respectively, using Bioprime kit (Invitrogen). Labeled DNA samples were coprecipitated with 50 mg of human Cot-1 DNA (Invitrogen), denatured, hybridized to BAC arrays for 48–72 h, washed, and counterstained with DAPI. Data processing Array CGH data image analyses were performed as described previously^{43 (link)}. Array probes missing in more than 50% of samples in the OncoBAC or scanning array data sets were excluded in the corresponding set. Array probes representing the same DNA sequence were averaged within each data set.