Bisulfite DNA sequencing on selected differentially methylated CG sites was performed to validate the results obtained from the Infinium HumanMethylation450 BeadChip arrays. One differentially methylated CG site in MX1 was selected for validation. Primers were designed using Sequenom's EpiDesigner online tool to include this CG site and an additional 20 CG sites in its vicinity. Primer quality was verified using Premier Biosoft’s Netprimer online tool (Sequenom, San Diego, CA). The following primers were used: forward 5’-TTTAGTGGATGTTATGTTTGGGGT-3’, reverse 5’-CACTACTACCTACCAAAACCCCTAAA-3’. PCR was performed on a Bio-Rad T100 (Bio-Rad, Hercules, CA) using ZymoTaq one-step master mix (Zymo, Orange, CA). The cycling conditions were as follows: 95°C for 10 min followed by 40 cycles of 95°C for 30 seconds, then 58.3°C for 40 seconds, then 72°C for 1 min, followed by 72°C for 7 min. Each PCR product was confirmed with a small aliquot by 2% agarose gel electrophoresis. The remainder of the PCR product was then purified and sequenced using an Applied Biosystems 3730 XL sequencer. Sequencing trace files were then used to calculate the percentage of cytosine methylation on each CG site using ESME software package (Epigenomics AG, Berlin).
Growth protocol
N/A (patient-derived samples)
Extracted molecule
genomic DNA
Extraction protocol
Fresh peripheral blood samples (25 ml) were collected and density gradient centrifugation (Ficoll) was used to collect PBMCs. LDGs were then isolated from PBMCs using indirect labeling and magnetic bead separation with the following antibodies: anti-CD3, anti-CD7, anti-CD19, anti-CD79b, anti-CD56, anti-MHCII, anti-CD86 and anti-CD235a. LDG purity was confirmed by flow cytometry using forward and side scatter profiles and was over 95% in all samples. Neutrophils were extracted from the granulocyte layer after Ficoll density gradient centrifugation, following previously described protocols
Label
Cy3 and Cy5
Label protocol
DNA was extracted from each sample using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA), then bisulfite-converted using the EZ DNA Methylation kit (Zymo Research, Irvine, CA) for DNA methylation studies.
Hybridization protocol
standard Illumina protocol
Scan protocol
standard Illumina protocol
Description
Neutrophil_Patient_10
Data processing
Following multiple quality control measures, probe intensity values were normalized and used to determine the average methylation level on each methylation site in each sample (beta value or β). Probes that include a genetic variant within the first 10bp region of the 3’ end of the probe, and probes that had a detection P value (detection above background) of ≥0.05 were excluded from the analysis. Differentially methylated sites between groups were determined, and defined as methylation sites with an absolute difference in beta value (delta beta or Δβ) of at least 0.1 and that remained significant (P<0.01) after correction for multiple testing using a Benjamini and Hochberg false discovery rate of 0.05 Unmethylated and methylated signal intensities in Amr_raw_methylation_data_SLE_neutrophils.txt
