Agilent 44k array

Copy number alterations were analyzed by microallelotyping using polymorphic dinucleotide microsatellite markers D5S642 and D5S2057, located 0.6 Mb centromeric and 1.8 Mb telomeric of ADAMTS19. In some cases where both markers were in homozygosis, we also analyzed D5S2098, located 5 Mb upstream of ADAMTS19. Primer sequences to amplify these markers were obtained from the Ensembl website [61 (link)]. PCR amplification was performed in presence of α-³²P-dCTP and resolved in vertical electrophoresis acrylamide-bisacrylamide gels. After electrophoresis, gels were dried and exposed to X-ray films. Loss of heterozygosity was assessed in heterozygous cases by the relative change in intensity in one of the bands when comparing the normal and tumor sample. aCGH was performed using Agilent 44K arrays, following the manufacturer’s protocol. Copy number alterations were analyzed using Agilent Genomic Workbench, with ADM-2 algorithm, threshold of 6, and Fuzzy Zero correction. Only alterations with a minimum of three consecutive probes were considered valid.

Methylation of histone 3 lysine residues were examined using ChIP as described [11 (link)]. ChIP-on-chip H3K27me3 and H3K9me2 was performed using amplified DNA generated from individual ChIP samples by PCR according to Affymetrix’s protocol. Sample fragmentation, labeling, hybridization, and data extraction were performed by the DNA Array Core facility at Johns Hopkins. GeneChip Human Tiling Promoter array (Affymetrix) was used for hybridizations. Analysis was performed using the model-based analysis of tiling array algorithm. Gene promoter-specific ChIP enrichment for the chromatin marks was compared to expression determined by Agilent 44K array.