Male rhesus monkeys (Macaca mulatta) were born between 2009 and 2010 and housed at the Laboratory of Comparative Ethology, part of the National Institute of Child Health and Human Development, at the National Institutes of Health Animal Center (Poolesville, MD). In the parental deprivation experiment monkeys were randomly divided into control mother-reared (MR) and surrogate peer-reared (SPR) groups at birth. Until weaning (7-8 months of age), the MR monkeys lived with their mothers in large social groups of 8-10 adult females, 1 adult male, and 3-5 same-aged peers. The SPR monkeys lived in individual cages with an inanimate surrogate mother, and continual visual, tactile, and olfactory access to same-aged peers, they had 2 hours of daily socialization periods with age-matched peers. At imposed weaning, all monkeys (MR and SPR) were relocated from their living conditions and placed in a mixed social group in a different building. Biological samples were collected at the 2 year routine medical check-up under chemical restrain (using ketamine).
Extracted molecule
genomic DNA
Extraction protocol
Peripheral blood was drawn into EDTA-coated tubes, and genomic DNA was extracted from 300 μl whole blood using the Wizard Genomic DNA Purification kit (Promega , Madison , WI , USA). Buccal epithelial cells were collected by rubbing cotton swabs on the palate and gum (3 swabs from each subject were isolated together). The dsDNA concentration was measured using the Qubit system (Life Technologies, Burlington, ON, Canada).
Label
Cy5
Label protocol
After the sonication step, 2 μg methylated DNA was immunoprecipitated with 5 μg of anti-5methyl-cytosine antibody (Eurogentec , Fremont , CA , USA) that is specific to DNA methylation, does not recognize hydroxymethylated DNA, and reveals changes in 5-methyl-cytosine exclusively. The DNA antibody complex was immunoprecipitated with protein G, and the methylated DNA was re-suspended in 0.25 ml of digestion buffer (50 mM TRisHCl pH = 8 , 10 mM EDTA , 0.5 % SDS) and treated with 40 μg of proteinase K overnight at 55°C. The input and bound fractions were phenol/chloroform-extracted and ethanol-precipitated The input and bound fractions were amplified using the Whole Genome Amplification Kit (Sigma-Aldrich , St. Luis , MO , USA) and then labeled for microarray hybridization with Cy3-dUTP and Cy5-dUTP, respectively, using the CGH Enzymatic Labeling Kit (Agilent Technologies , Mississauga , ON , Canada) in accordance with the manufacturer´s instructions.
gender: male protocol: MR chip antibody: none tissue: whole blood
Growth protocol
Male rhesus monkeys (Macaca mulatta) were born between 2009 and 2010 and housed at the Laboratory of Comparative Ethology, part of the National Institute of Child Health and Human Development, at the National Institutes of Health Animal Center (Poolesville, MD). In the parental deprivation experiment monkeys were randomly divided into control mother-reared (MR) and surrogate peer-reared (SPR) groups at birth. Until weaning (7-8 months of age), the MR monkeys lived with their mothers in large social groups of 8-10 adult females, 1 adult male, and 3-5 same-aged peers. The SPR monkeys lived in individual cages with an inanimate surrogate mother, and continual visual, tactile, and olfactory access to same-aged peers, they had 2 hours of daily socialization periods with age-matched peers. At imposed weaning, all monkeys (MR and SPR) were relocated from their living conditions and placed in a mixed social group in a different building. Biological samples were collected at the 2 year routine medical check-up under chemical restrain (using ketamine).
Extracted molecule
genomic DNA
Extraction protocol
Peripheral blood was drawn into EDTA-coated tubes, and genomic DNA was extracted from 300 μl whole blood using the Wizard Genomic DNA Purification kit (Promega , Madison , WI , USA). Buccal epithelial cells were collected by rubbing cotton swabs on the palate and gum (3 swabs from each subject were isolated together). The dsDNA concentration was measured using the Qubit system (Life Technologies, Burlington, ON, Canada).
Label
Cy3
Label protocol
After the sonication step, 2 μg methylated DNA was immunoprecipitated with 5 μg of anti-5methyl-cytosine antibody (Eurogentec , Fremont , CA , USA) that is specific to DNA methylation, does not recognize hydroxymethylated DNA, and reveals changes in 5-methyl-cytosine exclusively. The DNA antibody complex was immunoprecipitated with protein G, and the methylated DNA was re-suspended in 0.25 ml of digestion buffer (50 mM TRisHCl pH = 8 , 10 mM EDTA , 0.5 % SDS) and treated with 40 μg of proteinase K overnight at 55°C. The input and bound fractions were phenol/chloroform-extracted and ethanol-precipitated The input and bound fractions were amplified using the Whole Genome Amplification Kit (Sigma-Aldrich , St. Luis , MO , USA) and then labeled for microarray hybridization with Cy3-dUTP and Cy5-dUTP, respectively, using the CGH Enzymatic Labeling Kit (Agilent Technologies , Mississauga , ON , Canada) in accordance with the manufacturer´s instructions.
Hybridization protocol
All the steps of hybridization, washing, scanning and feature extraction were performed following the Agilent protocols for chip-on-chip analysis (MeDIP).
Scan protocol
Scanning was performed using the Agilent Technologies Scanner G2505B. Probe intensities were extracted from scanned microarray images using Agilent's Feature Extraction 10.7.3.1 Image Analysis Software
Data processing
The extracted intensities were then analyzed using the R software environment for statistical computing (R Development Core Team, 2007) (http://www.r-project.org/). Log-ratios of the bound (Cy5) and input (Cy3) microarray channel intensities were computed for each microarray. Microarray quality was assessed using plots generated by the feature extraction software in addition to correlation matrix visualizations. Microarrays were normalized using quantile-normalization 8. Estimates of DNA methylation levels based on microarray probe intensities were obtained using a Bayesian deconvolution algorithm. Differential methylation between groups was determined in two stages. The first stage used linear models implemented in the ‘limma’ package 10 of Bioconductor 11 to compute a modified t-statistic at the individual probe level. As in typical microarray studies, the number of sample profiles is small so there is little information per probe from which to estimate probe variance. Hence, the modified t-statistic for each probe makes use of variance estimates derived from the other probes on the microarray. Correlation between technical replicates was modeled as a random effect using the “block” variable. An individual probe was called differentially methylated if the significance of its t-statistic was at most 0.05 (uncorrected for multiple testing) and the associated difference of log-normalized means between the groups was at least 0.5. In the second stage, differential statistics per promoter were derived from the t-statistics of the probes within the promoter. In particular, the Wilcoxon rank-sum test was used to identify enrichment for probes within the promoter having large positive or large negative t-statistics. The resulting promoter p-values were adjusted for multiple testing by calculating false discovery rates using the Benjamini-Hochberg algorithm. A promoter was called differentially methylated if its false discovery rate was at most 0.2 and it contained at least one probe called differentially methylated, as defined above.
