Nimblescan software

Briefly, RNA from tissue samples (three melanomas and three normal tissues) was used to synthesize double-stranded cDNA, and double-stranded cDNA was labeled and hybridized to the 2.0 microRNA Expression Microarray (Affymetrix GeneChip Human Gene 2.0 ST Array, Rockville, MD, USA). Raw data were extracted as pair files using NimbleScan software (version 2.5; Roche NimbleGen, Inc., Madison, WI, USA). NimbleScan software’s implementation of RMA offers quartile normalization and background correction. Differentially expressed genes were identified through the random variance model. The AP value was calculated using the paired t-test. The threshold set for up- and downregulated genes was a fold change > 2.0 and a P-value < 0.05. Hierarchical clustering was performed based on differentially expressed miRNAs using Cluster_Treeview software from Stanford University (Palo Alto, CA, USA).

The VEEV cDNA samples were fluorescently labeled and hybridized to VEEV SNP arrays as described previously [20 (link)]. Briefly, fluorescent labeling of samples was performed using the NimbleGen One-Color DNA Labeling Kit (Roche). One μg VEEV cDNA was added to Cy-3 labeled random primers, followed by isothermal amplification at 37°C using Klenow polymerase. Labeled DNA was purified via isopropanol precipitation and resuspended in water for microarray hybridization. DNA samples were prepared for hybridization using the NimbleGen Hybridization Kit LS (Roche). Three μg of labeled DNA was hybridized to each array, incubating for 40–45 hours at 42°C. Arrays were washed using the NimbleGen Wash Buffer Kit (Roche). The fluorescent signal on the array was scanned using a 2 μm Roche MS200 fluorescent scanner. Array feature intensities were generated using the NimbleScan software available from Roche NimbleGen.

DNA samples of three MZ twin sets (#1/2, 9/52, and 24/57; see Table 1) were sonicated and then immunoprecipitated with a monoclonal antibody that specifically recognizes 5-methylcytidine (Roche NimbleGen, Madison, WI, USA). DNA fragments were converted into PCR-amplifiable OmniPlex™ Library molecules flanked by universal primer sites and the library amplified by PCR using universal primers and a limited number of cycles. Immunoprecipitated and reference DNA were tagged, respectively, with cyanine-5 (Cy5) and cyanine-3 (Cy3)-labeled random 9-mers and then hybridized by the NimbleGen Array Hybridization Kit (Roche NimbleGen, Madison, WI, USA).
A four-plex array was custom-designed to include 998 X chromosome and 18,086 randomly selected autosomal chromosome promoter sites (Roche NimbleGen, Madison, WI, USA) and samples analyzed following the manufacturers protocols. First, Nimblescan software (Roche NimbleGen, Madison, WI, USA) was utilized for DNA methylation data analysis using a threshold p-value of 0.05 equivalent to 1.31 based on the Gaussian distribution of data. Second, exclusive elements corresponding to specific microarray probes were identified in affected and healthy subjects and peaks found only in either group were selected for further analysis. Third, elements of interest were inserted into the UCSC Genome Browser (GRCh36/hg19) to identify corresponding genes.

Datasets corresponding to the histone enrichment values in both the immunoprecipitated (IP) and the input samples were processed to obtain Ratio.GFF files (log₂ IP/input). All the samples passed the QC metrics provided by Roche NimbleGen (Roche). The Ratio.GFF files from the triplicates for each experiment were merged to obtain average values for histone modification enrichment. These were then converted to wiggle files for visualization in the IGV genome browser. Initial peak calling was performed using NimbleScan software (Roche), and initial calls were further filtered to obtain significant peaks (log₂ ratio > 1 and the false discovery rate (FDR) < 5%). The filtered peaks were subsequently mapped to overlapping features 5000 bp upstream and 1000 bp downstream of the nearest transcription start site (TSS), and a peak report was generated. The promoter information and coordinates were used for downstream analysis.