Massarray analyzer 4

The rs12415800 variant in the SIRT1 gene was genotyped. It is located upstream of the 5′ end of the SIRT1 gene and is the first reported gene polymorphism associated with major depression in the Chinese population through a large-sample GWAS study [CONVERGE consortium (24 (link))]. The gene extraction and genotyping method used in this study is consistent with our previously published paper (25 (link)). In brief, DNA was extracted from peripheral venous blood using the Trizol protocol, and genotyping was performed using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry on the MassARRAY Analyzer 4 platform (Sequenom, San Diego, CA, United States). Probes and primers were designed using the My-Sequenom online software Assay Design Suite v2.0.

A total of 12-16 nl of each iPLEX® reaction product were transferred onto a SpectroCHIP® II G96 (SEQUENOM Inc., San Diego, CA, USA) using SEQUENOM® MassARRAY® Nanodispenser (SEQUENOM Inc., San Diego, CA, USA). SpectroCHIP® analysis was carried out by SEQUENOM® MassArray® Analyzer 4 and the SpectroAcquire software Version 4.0 (SEQUENOM Inc., San Diego, CA, USA). Finally data analysis for genotype determination was done using the MassARRAY® Typer software version 4.0 (SEQUENOM Inc., San Diego, CA, USA). In order to confirm the genotypes obtained, randomly selected samples (5 each for case and control cohorts) from each genotype (n = 240) were validated by Sanger Sequencing to ensure accuracy of genotyping results. In all cases, the Sanger Sequencing confirmed the genotyping obtained using MassARRAY.

Genomic DNA was isolated from the purified cell pellets using the QIAmp DNA Mini Kit (Qiagen) according to the manufacturer’s protocol. DNA from whole blood was extracted by the salting-out method using 10 M ammonium acetate. The DNA was precipitated in isopropanol, washed in 70% ethanol, and finally resuspended in 1X TE buffer. The purity and concentrations of the DNA samples were measured by NanoDrop ND-1000 spectrophotometry. 500 ng of genomic DNA was treated with sodium bisulfite using the EZ DNA Methylation Kit (Zymo Research Corporation) according to the manufacturer’s instructions. DNA methylation analysis was performed using the Infinium Human Methylation 450 K BeadChip (Illumina). Quantitative analysis of DNA methylation was verified using Sequenom’s EpiTYPER T Complete Reagent Set and MassARRAY Analyzer 4 (Sequenom). DNA was amplified using bisulfite-converted DNA, Hot Start DNA Polymerase (Solis BioDyne) and specific primers, according to the EpiTYPER protocol. Primers were designed with Sequenom’s EpiDesigner program and are listed in Supplementary Table S4.

Genomic DNA was extracted from venous blood leukocytes using the EZ1 DNA Blood 350 μL kit (Qiagen) according to the manufacturer's instructions for genotyping. Seven SNPs related to NAFLD relevant traits, including rs2071518 in cellular communication network factor 3 (CCN3), rs12409877 in leptin receptor (LEPR), rs10770141 in tyrosine hydroxylase (TH), rs4430796 in hepatocyte nuclear factor 1-beta (HNF1B), rs2292354 in G protein-coupled receptor kinase interactor 2 (GIT2), rs5186 in angiotensin II receptor type 1 (AGTR1) and rs2206277 in transcription factor AP-2 beta (TFAP2B) from NCBI database of SNP database (www.ncbi.nlm.nih.gov/SNP) were analyzed, and genotyped by matrix-assisted laser desorption/ionization time-off light mass spectrometer in MassARRAY Analyzer 4 platforms (Sequenom, San Diego, CA). Probes and primers were determined with online Assay Design Suite version 2.0 software. Polymerase chain reaction was performed according to the instructions of the manufacturers. More detailed information about primers and polymerase chain reaction conditions is available upon request.

Genomic DNA was extracted from peripheral blood leukocytes using QIAamp DNA Blood Mini Kit (QIAGEN, Germany), following the manufacturer's protocol. SNPs in SPP1 (rs28357094, rs11730582, and rs17524488) and LTPB4 (rs2303729, rs1131620, rs1051303, and rs10880) were genotyped by Sequenom MassARRAY iPLEX platform (Sequenom, San Diego, CA, USA), following standard protocols and using primers designed in Sequenom MassARRAY Design 3.1. To enhance differences between alleles, targets were amplified by PCR, reacted with shrimp alkaline phosphatase, and extended with iPLEX primers. After purification on clean resin, products were spotted onto a 384-well SpectroCHIP using MassARRAY RS 1000, analyzed by mass spectrometry on a MassARRAY Analyzer 4 (Sequenom), and called in MassArray Typer 4.0 (Sequenom). Ten percent of samples were duplicated for quality control, and failed reactions were re-genotyped by Sanger sequencing.

Genomic DNA was extracted from venous blood leukocytes by the standard phenol-chloroform method. Totally, eight SNPs were selected from the literature and the National Center for Biotechnology Information dbSNP database (http://www.ncbi.nlm.nih.gov/SNP) for Genotyping by a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer using the MassARRAY^® Analyzer 4 platform (Sequenom, CA, USA). The SNPs information included in the final analysis is listed in Table 1. The minor allele frequency (MAF) of SNPs in our present study was comparable to that in East Asian population reported in the 1000 Genomes Project [21 (link)]. In order to ensure the reliability of genotyping quality, quality control was carried out at both an individual level and a SNP level [22 (link)]. At the individual level, subjects with incomplete information were excluded. In addition, individuals with a call rate of less than 0.8 were also excluded. SNPs that violated Hardy-Weinberg equilibrium (HWE<0.05) were removed at the SNP level. Moreover, no template controls (>1%) were called blind to their status in the genotyping process and each SNP was re-genotyped in at least 5% random DNA samples.