cell type: CD4+T-cells disease status: healthy control
Treatment protocol
Peripheral blood was collected from patient samples and controls.
Growth protocol
Twelve female lupus patients and 12 female controls were studied. The mean age was 42.83±3.61 in patients and 43.75±3.40 in controls (mean±SEM).
Extracted molecule
genomic DNA
Extraction protocol
CD4+ T lymphocytes were obtained via Ficoll/Paque PBMC enrichment (GE Healthcare Life Sciences, Piscataway, NJ) followed by CD4+ isolation using a MACS magnetic bead CD4+ T-cell isolation kit (Miltenyi Biosystems, Auburn, CA). Whole genomic DNA was then prepared using a Qiagen DNEasy kit (Qiagen, Germantown, MD) and bisulfite-treated using a Zymo EZ DNA Methylation Kit (Zymo, Orange, CA).
Label
biotin (C and G nucleotides) or dinitrophenyl (A and T nucleotides)
Label protocol
Bisulfite-converted patient and control DNA samples were prepared and quantified using a NanoDrop scanning spectrophotometer (Thermo, Wilmington, DE). For each sample, 500ng of whole-genome bisulfite-converted DNA was denatured, fragmented, and amplified using Illumina-supplied reagents according to manufacturer instructions. Following precipitation, DNA was hybridized to Illumina Infinium HumanMethylation27 arrays, containing 50-mer oligonucleotides designed to hybridize either methylated (N-CG-N following bisulfite conversion) or unmethylated (N-TG-N following bisulfite conversion) cytosine on each CG pair interrogated, coupled to beads mounted on glass slides. Microarrays were washed under high stringency, labeled with biotin (C and G nucleotides) or dinitrophenyl (A and T nucleotides), and scanned with an Illumina iScan.
Hybridization protocol
DNA was hybridized to Illumina Infinium HumanMethylation27 arrays, containing 50-mer oligonucleotides designed to hybridize either methylated (N-CG-N following bisulfite conversion) or unmethylated (N-TG-N following bisulfite conversion) cytosine on each CG pair interrogated, coupled to beads mounted on glass slides.
Scan protocol
Illumina iScan
Description
Healthy
Data processing
The relative level of methylation for each CG site was calculated as the ratio of methylated-probe signal to total locus signal intensity, and defined within 0 to 1 range, exported from the Illumina BeadStudio software package. The data were normalized as described previously, (Dozmorov I, Knowlton N, Tang Y, Shields A, Pathipvanich P, Jarvis JN, et al. Hypervariable genes--experimental error or hidden dynamics. Nucleic Acids Res 2004; Dozmorov I, Lefkovits I. Internal standard-based analysis of microarray data. Part 1: analysis of differential gene expressions. Nucleic Acids Res 2009) using the variability of areas with low methylation level as a reference point. In order to find genes with methylation level above the level of technical noise, a frequency histogram of raw methylation signal was determined for each array. The histogram yielded a right-skewed unimodal distribution curve with a mode of approximately 0.013. A normal distribution curve representing the variability of the data around zero was then fitted around the mode, mirroring the Gaussian profile of the left part of the histogram. Its parameters were then defined (mean, SD) and the data were normalized to the standard deviation of the noise after subtraction of the mean. The data were then Log10-transformed and adjusted to each other by robust linear regression under the assumption that the methylation of most genes does not change. The data were then filtered to remove genes with a methylation level value less than 3.0, equivalent to setting a threshold at three standard deviations above the noise level. Genes with methylation differential below the noise level under all experimental conditions (about 1,600) were excluded from consideration, as their methylation cannot be reliably assessed.
