METHODS

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 5 × volume of PBS, 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.

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).

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).

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.

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 with a 5 × volume of PBS, 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.

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 with a 5 × volume of PBS, 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.

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).

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.

TBS was performed using Illumina HiSeq 2500 with the read length of 125 bp (paired-end). Genomic regions of interest were enriched using custom-designed oligonucleotide probe sets. Three probe sets have been designed so far: CDMV (common DNA methylation variation sites) version 1 was designed to cover ~341,000 CpGs with higher inter-individual variability and associated with the expression of neighboring genes (eQTM) identified in CD4T and monocytes, CDMV version 2 was designed to efficiently cover ~5 million CDMV CpGs in CD4T, monocytes, and neutrophils, and CDMV version 3 was designed to efficiently cover ~1.5 million CDMV or eQTM CpGs identified in CD4T, monocytes, neutrophils, PBMCs, and whole-blood. Sequence reads obtained from each version of the probe-set were mapped onto GRCh37d5 using NovoAlign. The beta value (ratio of the number of methylated reads to the total number of reads mapped to each CpG site) was used to represent the DNA methylation level.

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.

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 dataset (monocytes, CD4+ T cells, neutrophils, and PBMC). In these analyses, we defined cis-proximity as ± 5kb in distance. Simple linear regression was fitted in each analysis.
For the analyses of monocytes, CD4T, and neutrophils, same datasets of genotype, DNA methylation, and gene expression on iMETHYL browser were used (see table 1 in STATISTICS).

[Mar. 24 2020 updated] Gene expression data was generated through a new pipeline using STAR and RSEM, and TPM values were used in the QTL analyses. For QTL analyses of monocytes, CD4T, and neutrophils, only CpGs with call rate ≥ 50% across cell types were used.