gender: male protocol: stress developmental stage: 1month chip antibody: anti-5-methylcytidine (33D3) antibody (Eurogentec) cell type: CD3+ T lymphocyte
Growth protocol
Male and female rhesus monkeys (Macaca mulatta) were born between 2009 and 2011 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 maternal deprivation experiments monkeys were randomly divided into control (mother-reared, MR) and stress (surrogate peer-reared, SPR) groups at birth. Until imposed 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. After imposed weaning, all monkeys (MR and SPR) were relocated from their living conditions and placed in a mixed social group in a different building where they lived up through 3 years of age.
Extracted molecule
genomic DNA
Extraction protocol
Peripheral blood was drawn into EDTA-coated tubes. PBMCs were isolated through centrifugation with Ficoll-Paque (GE Healthcare, Burnaby, BC, Canada), and T cells were isolated from the PBMCs using CD3+ Dynabeads (Life Technologies, Burlington, ON, Canada). The CD3+ T cell DNA was extracted using the Wizard Genomic DNA Purification kit (Promega; Madison; WI; USA). The dsDNA concentration was measured using the Qubit system (Life Technologies, Burlington, ON, Canada). The 1 month samples were pooled DNA (male and female separately) from 6 control and 5 stress subjects sampled twice, i.e., at postnatal days 14 and day 30, because of low DNA yield in individual samples. The DNA pooling procedure was applied for all time points, using the same amount of dsDNA from each individual sample, creating 4 different groups at the actual developmental stage: male control, male stress, female control, female stress.
Label
Cy5
Label protocol
After the sonication step, methylated DNA was immunoprecipitated with 10 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: stress developmental stage: 1month chip antibody: none cell type: CD3+ T lymphocyte
Growth protocol
Male and female rhesus monkeys (Macaca mulatta) were born between 2009 and 2011 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 maternal deprivation experiments monkeys were randomly divided into control (mother-reared, MR) and stress (surrogate peer-reared, SPR) groups at birth. Until imposed 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. After imposed weaning, all monkeys (MR and SPR) were relocated from their living conditions and placed in a mixed social group in a different building where they lived up through 3 years of age.
Extracted molecule
genomic DNA
Extraction protocol
Peripheral blood was drawn into EDTA-coated tubes. PBMCs were isolated through centrifugation with Ficoll-Paque (GE Healthcare, Burnaby, BC, Canada), and T cells were isolated from the PBMCs using CD3+ Dynabeads (Life Technologies, Burlington, ON, Canada). The CD3+ T cell DNA was extracted using the Wizard Genomic DNA Purification kit (Promega; Madison; WI; USA). The dsDNA concentration was measured using the Qubit system (Life Technologies, Burlington, ON, Canada). The 1 month samples were pooled DNA (male and female separately) from 6 control and 5 stress subjects sampled twice, i.e., at postnatal days 14 and day 30, because of low DNA yield in individual samples. The DNA pooling procedure was applied for all time points, using the same amount of dsDNA from each individual sample, creating 4 different groups at the actual developmental stage: male control, male stress, female control, female stress.
Label
Cy3
Label protocol
After the sonication step, methylated DNA was immunoprecipitated with 10 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.
