DNA were isolated from these selected samples using the DNeasy kit® (Qiagen, Valencia, CA) and Invitrogen PureLink Mini Kit® (Life Technologies Corporation, Carlsbad, CA), respectively, and stored at -80C. DNA were quantified by using a NanoDrop® (ThermoScientific, Wilmington, MA),
Label
cy3
Label protocol
The high molecular weight genomic DNA was sonicated to generate 200-500bp sized DNA fragments using a Bioruptor sonicator® (Diagenode, Denville, NJ) using cycle conditions of 30 sec on/90 sec off for a total duration of 12.5 minutes. Methylated DNA molecules were enriched by binding to the MBD2B/MBD3L1 complex using the MethylCollector Ultra kit® (Active Motif, Carlsbad, CA) according to the manufacturer’s protocol. Input and enriched methylated-DNA fragments from the same sample were whole genome amplified (WGA) using a WGA2 kit® (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s protocol
cohort: CNTL replicate: 4 fraction: methylated DNA
Treatment protocol
Vehicle control by oral gavage for 2 years
Extracted molecule
genomic DNA
Extraction protocol
DNA were isolated from these selected samples using the DNeasy kit® (Qiagen, Valencia, CA) and Invitrogen PureLink Mini Kit® (Life Technologies Corporation, Carlsbad, CA), respectively, and stored at -80C. DNA were quantified by using a NanoDrop® (ThermoScientific, Wilmington, MA),
Label
cy5
Label protocol
The high molecular weight genomic DNA was sonicated to generate 200-500bp sized DNA fragments using a Bioruptor sonicator® (Diagenode, Denville, NJ) using cycle conditions of 30 sec on/90 sec off for a total duration of 12.5 minutes. Methylated DNA molecules were enriched by binding to the MBD2B/MBD3L1 complex using the MethylCollector Ultra kit® (Active Motif, Carlsbad, CA) according to the manufacturer’s protocol. Input and enriched methylated-DNA fragments from the same sample were whole genome amplified (WGA) using a WGA2 kit® (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s protocol
Hybridization protocol
The labeling of WGA IP and input DNA, microarray hybridization, and scanning were performed at the Florida State University genomics core laboratory following the described protocol (Roche NimbleGen (2010)).
Scan protocol
Data were extracted from scanned images using NimbleScan 2.4 extraction software (NimbleGen Systems, Inc., Madison, WI)
Description
Methylation Signal for CNTL 4
Data processing
The probe-level log2(Cy5/Cy3) values were normalized using Lowess curve fitting conditioning on GC content as described in BMC Bioinformatics 2009, 10:173. These signal values were further quantile normalized to adjust for between sample variations. DETAILS: The GC Lowess method utilizes both 'Cy3' and 'Cy5' values as following: * Classify probes into bins according to CG content (proportion of number of C and G nucleotides in probe sequence) * For each CG content bin (k), we fit lowess regression to predict log2(Cy5/Cy3)_k value using log2(sqrt(Cy3*Cy5))_k value * Compute normalized value = sigma_bar *(observed log2(Cy5/Cy3)_k - predicted log2(Cy5/Cy3)_k)/sigma_k, where sigma_k is mean absolute deviation of (observed log2(Cy5/Cy3)_k - predicted log2(Cy5/Cy3)_k) and sigma_bar is geometric mean of all sigma_k's. This normalization corrects for both dye bias and CG bias simultaneously.
Genome-wide promoter DNA Methylation Profiling of Hepatocellular Carcinomas Arising Either Spontaneously or due to Chronic Exposure to Ginkgo biloba Extract (GBE) in B6C3F1/N Mice [Methylation]
