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MHC multimers of a given specificity were then formed using probe-tagged streptavidin molecules, providing each pMHC complex with a probe that allows binding to a specific site on the microarray.
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Initially, we have compared the overall methylation level in all 27,578 CpG sites on the microarray with the methylation levels of those CpG sites which have been assigned for histone modifications.
This is in line with the fact that the senescence-associated CpG sites displayed a much higher median methylation level (47.2%) compared to all CpG sites on the microarray.
We observed that the number of dmCpG sites for a gene was not dependent on the total number of CpG sites present on the microarray chip for the same gene (Pearson's correlation = 0.55, and Fig. S1, Supporting Information).
Hierarchical clustering of 85 CpG sites represented on the microarray that are associated with the four HOX clusters revealed methylation changes that correlate with different anatomical sites (Figure S4).
This might be due to the bias for promoter regions within the 27,578 CpG sites represented on the microarray whereas it has been shown, that hypo-methylation upon aging occurs predominantly in specific interspersed repetitive sequences (IRSs) [42].
Alternatively we have used the Bayesian method Significance Analysis of Microarray (SAM) to estimate the false discovery rate (FDR): 8,370 of the 27,578 CpG sites represented on the microarray were differentially methylated (FDR = 5%).
The raw binding intensity (R ij ) of each probe was defined as F ij / B ij, where F ij and B ij are the median values of foreground and background signals of the probe at site (i, j) on the microarray, respectively.
Break sites were randomized by randomly selecting an equal number of positions on the microarray.
Start sites for randomly selected regions were constrained to start sites of probes represented on the microarrays to control for bias introduced by probe design/selection on the microarrays.
For microarrays, this problem is multiplied by the number of genes on the microarray itself.
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