Genomic workbench standard edition 5

Two PARK4 blood samples were assessed for copy number variation using a SurePrint G3 Human CGH (comparative genomic hybridization) microarray 2×400 K design 021850 (Agilent Technologies). Agilent's labeling kit was used, and all procedures were carried out according to the manufacturer's instructions. Scanning was performed on an Agilent microarray scanner and raw data were processed by Feature Extraction 9.5. Deleted and amplified regions were determined on Agilent's Genomic Workbench Standard Edition 5.0.14. A minimum of four consecutive probes had to be affected to make a decision. The aberration detection threshold of the ADM-2 algorithm was set to 5.9.

Ten nM BrU-labelled TUG1 RNA (2,133–2,910 nucleotides) was transfected into GSCs using Lipofectamine 2000 (Life Technologies). After 72 h of transfection, GSCs were cross-linked with 0.5% formaldehyde, and chromatin fractions were extracted and homogenized. Chromatin and BrU-labelled TUG1 RNA mixtures were immunoprecipitated using an anti-BrU antibody and Dynabeads Protein G. Purified chromatin was eluted to yield DNA, which was then subjected to microarray analysis or ChIP-qPCR analysis.
Equivalent amounts of DNA, which was extracted after immunoprecipitation for BrU-labelled TUG1 RNA, and total input DNA were amplified in parallel using a random primer method with the GenomePlex Complete Whole Genome Amplification Kit (15 cycles), according to the manufacturer's instructions (Sigma-Aldrich). Amplified products were labelled with Cy5 for immunoprecipitated DNA samples and Cy3 for input DNA using the BioPrime Array CGH Genomic Labeling System (Life Technologies). A total of 4 μg of labelled DNA was then hybridized to the human promoter ChIP-chip microarray (G4874A, Agilent Technologies) for 48 h at 65 °C. Data were extracted from scanned images using the Feature Extraction software, version 10.7.3.1 (Agilent Technologies). The text files were then imported into Genomic Workbench, standard edition 5.0.14 (Agilent Technologies), for analysis.