METHODS

Protocols for the cell isolations

Isolation of Monocytes and CD4+ T cells from PBMCs (N = 102)

  • PBMCs were stained with CD14-FITC, CD16-PE, CD3-PE/Cy7, and CD4-APC antibodies (5 µL / 500 µL cell suspension; Sony Biotechnology, Inc., Tokyo, Japan) and incubated for 20 min at 4°C. After washing with a rinsing solution (PBS containing 2 mM EDTA (pH 7.2) and 5 w/v% new born calf serum), CD14++/CD16- monocytes and CD3+/CD4+ T cells were immediately sorted using a CellSorter SH800 (Sony Biotechnology) from the monocyte- or lymphocyte-containing gate determined by light-scatter density-plot.

Protocol for the isolation of Neutrophils from Peripheral blood (N = 94)

  • Polymorph nuclear cells were stained with CD16-PE antibody (5 µL / 500 µL cell suspension; Sony Biotechnology) and incubated for 20 min at 4°C. After washing with rinsing solution (PBS containing 2 mM EDTA (pH 7.2) and 5 w/v% new born calf serum), CD16+ neutrophils were immediately sorted using a CellSorter SH800 (Sony Biotechnology).

Protocol for the isolation of leukocytes (N = 20)

  • 1 mL of whole blood was added into 30 mL of red blood cells lysis buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA), and the tube was inverted several times. After incubating for 10 min at room temperature, the cells were washed with rinsing solution (PBS containing 2 mM EDTA (pH 7.2) and 5 w/v% new born calf serum).

Protocol for the isolation of PBMCs and polymorph nuclear cells (N = 20)

  • PBMCs and polymorph nuclear cells were separated from 7 mL of whole blood by Mono-Poly Resolving Medium (DS Pharma Biomedical Co., Ltd.) according to the manual.

Protocol for the isolation of monocytes and natural killer cells (N = 20)

  • PBMCs were stained with CD14-FITC, CD16-PE and CD56-APC antibodies (5 µL / 500 µL cell suspension; Sony Biotechnology) and incubated for 20 min at 4°C. After washing wit rinsing solution (PBS containing 2 mM EDTA (pH 7.2) and 5 w/v% new born calf serum), CD14++/CD16- T cells and CD56+ NK cells were immediately sorted using a Cell Sorter SH800 (Sony Biotechnology) from the monocyte- or lymphocyte-containing gate determined by light-scatter density-plot.

Protocol for the isolation of CD4+ T cells, CD8+ T cells and B cells (N = 20)

  • PBMCs were stained with CD8-FITC, CD19-PE, CD3-PE/Cy7, and CD4-APC antibodies (5 µL / 500 µL cell suspension; Sony Biotechnology) and incubated for 20 min at 4°C. After washing withrinsing solution (PBS containing 2 mM EDTA (pH 7.2) and 5 w/v% new born calf serum)S, CD3+/CD4+ T cells, CD3+/CD8+ T cells and CD3-/CD19+ B cells were immediately sorted using a CellSorter SH800 (Sony Biotechnology) from the lymphocyte-containing gate determined by light-scatter density-plot.

Sequencing

Whole-genome sequencing (WGS)

  • WGS was performed using Illumina HiSeq 2500 or HiSeq X with the read length of 162 bp or 150 bp (paired-end). Sequence reads were mapped onto the human reference genome GRCh37 using BWA-MEM (ver. 0.7.5a-r405) and valiant calling was performed using GATK (ver. 2.5-2).

Whole-genome bisulfite sequencing (WGBS)

  • WGBS was performed using Illumina HiSeq 2500 or HiSeq X with the read length of 125 bp or 150 bp (paired-end). Sequence reads were mapped onto GRCh37d5 using NovoAlign (ver. 3.02.08). Beta value, that is the ratio of methylated-read number to the total read number mapped onto the CpG site, was used to represent the DNA methylation level.

RNA sequencing

  • RNA-Seq was performed using Illumina HiSeq 2500 with the read length of 125 bp (single-end). Sequence reads were mapped onto GRCh37 using TopHat (ver. 2.0.13) For the gene annotation, Human GENCODE Gene Set (release 19) was used. log10(FPKM + 0.1) was applied to represent the gene expression level.

QTL analyses

  • Associations between genotype and gene expression (cis-eQTL), DNA methylation and gene expression(cis-eQTM), and genotype and DNA methylation (cis-mQTL) were tested for each cell-type (monocytes, CD4+ T cells, and neutrophils). In these analyses, we defined cis-proximity as <1 Mb in distance. Simple linear regression was fitted in each analysis.
  • Same dataset of genotype, DNA methylation, and gene expression for each cell-type were used as the iMETHYL browser (see table 1 in STATISTICS).
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