Sequencher software version 4

Sequence data were assembled and analyzed for the presence of SNPs and indels using SEQUENCHER software version 4.5 (Gene Codes Corporation, Ann Arbor, MI, USA). Assembled sequences were aligned with each other and the published GenBank sequence (accession: EF064741.1) using SEQUENCHER, to identify polymorphisms. The GenBank NCBI SNP database (dbSNP) was searched for all reported SNPs in the CXCR6 gene to determine whether polymorphisms detected in this study had been previously reported. The first nucleotide of the CXCR6 translational start site was designated as +1 and the nucleotide immediately upstream from that as -1.

For each MA case, tumor genomic DNA was isolated from FFPE tissue blocks using the QIAamp DNA FFPE Tissue Kit (Qiagen, Venlo, Netherlands), according to the manufacturer's protocol. Commercially available male human genomic DNA (G1471; Promega, Madison, WI) served as a wild type BRAF exon 15 control, while genomic DNA isolated from SK-MEL-5 cells (HTB-70; ATCC, Manassas, VA) using the DNeasy Blood & Tissue Kit (Qiagen) served as a positive BRAF V600E control. BRAF exon 15 was PCR amplified from 50 nanograms of genomic DNA using flanking sequence-specific primers (forward: 5′-M13-TTTGTGAATACTGGGAACTATGAAA-3′; and, reverse: 5′-TCATCCTAACACATTTCAAGCC-3′) and HotStarTaq DNA polymerase (Qiagen). The PCR conditions were as follows: initial denaturation at 95°C for 15 minutes; 45 cycles of 94°C for 60 seconds, 50°C for 60 seconds, and 72°C for 60 seconds; and, final extension at 72°C for 7 minutes. After confirming the expected amplicon size by standard agarose gel electrophoresis, all PCR products were incubated with ExoSAP-IT (Affymetrix, Santa Clara, CA) prior to bi-directional Sanger sequencing by the University of Michigan DNA Sequencing Core using the M13 forward and BRAF exon 15 PCR reverse primers. The resulting chromatograms were analyzed with Sequencher software, version 4.5 (Gene Codes, Ann Arbor, MI), and compared to reference sequence for BRAF exon 15 (NM_004333.4).

Frontal lobe tissue of subjects with APOEε3/ε4 genotypes were selected for this experiment. Bisulfite converted genomic DNA was PCR amplified by bisulfite-specific primers (Supplementary Table 1) using HotStarTaq DNA PCR master mix (Qiagen) with a profile of 95°C for 15min, followed by 30 cycles of 95°C for 20 s, 50°C for 20 s, and 72°C for 1 min, ending with 72°C for 5 min. PCR fragments were purified using the NucleoSpin Gel and PCR Clean-Up kit (Clontech, Mountain View, CA) and were cloned into Xhol and Hindlll double-digested pGL4 vector (Promega, Madison, WI) using In-Fusion HD Cloning Plus kit (Clontech). The recombinant DNA was then transformed into Stellar competent cells (Clontech). A minimum of 19 clones from each sample were randomly selected and their inserts were PCR amplified using pGL4 vector-specific forward and reverse primers (Supplementary Table 1). PCR products were treated with ExoSAP-IT (Affymetrix, Santa Clara, CA) overnight at 37°C to remove the residual primers and deoxyribonucleotide triphosphates prior to sequencing. PCR fragments were sequenced by the pGL4_R1 primer (Supplementary Table 1) using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) on a 3130xL Genetic Analyzer (Applied Biosystems). Sequences were analyzed by Sequencher software version 4.9 (Gene Codes Corp, Ann Arbor, Michigan).