Sequence specific primers

Genomic DNA was extracted from whole blood or buffy coats (QIAamp blood kit; Qiagen). HLA genotyping relied on a combination of PCR-based techniques, involving sequence-specific primers (Invitrogen) and sequence-specific oligonucleotide probes (Innogenetics), as described previously [74 (link)]. Ambiguities were resolved by direct sequencing of three exons in each gene, using kits (Abbott Molecular, Inc.) designed for capillary electrophoresis and the ABI 3130xl DNA Analyzer (Applied Biosystems).
SNP genotyping with the Illumina ImmunoChip was processed at a genomics core facility (University of Alabama at Birmingham); SNP alleles were inferred using the joint calling and haplotype phasing algorithm implemented in BEAGLECALL[75 (link)]. We completed a series of data cleaning and quality control procedures for SNPs in the xMHC region, excluding SNPs based on the following criteria: (i) duplication, (ii) missingness (call rate<98.5%), (iii) minor allele frequency <0.025 in SCs and <0.015 in SPs and (iv) deviation from Hardy–Weinberg equilibrium (P<10⁻⁶). Data processing and quality control procedures have been described previously for this data set[46 (link)].

Total RNA was isolated from human frozen tissues using the mirVana miRNA isolation kit (Ambion, Austin, Texas, USA) according to the manufacturer's instructions. The concentration and purity of RNA were spectrophotometrically determined by measuring the optical density (A260/280>2.0, A260/230>1.8) using a NanoDrop ND-2000 spectrophotometer (Thermo Scientific Wilmington, DE, USA). cDNA was synthesized according to the TaqMan microRNA Assay protocol (Applied Biosystems) using 1 μg of total RNA as a template. qPCR analyses were conducted to quantify mRNA expression using SYBR Premix (Applied Biosystems) and GAPDH as an internal control. The sequences of the primers were as follows: FZD8 forward, 5′-GATGGGATTGCACGGTTTGG-3′, FZD8 reverse, 5′-ACCCGTATTTACGTGGGGTG-3′; GAPDHforward,5′-AATCCCATCACCATCTTCCA-3′, GAPDH reverse 5′-TGGACTCCACGACGTACTCA-3′. The TaqMan MicroRNA Assay kit (Applied Biosystems) was used to measure the levels of miR-375 after reverse transcribing with sequence-specific primers (Applied Biosystems), and U6 small nuclear RNA was used as an internal control. Real-time PCR was performed on an Applied Biosystems 7500 Sequence Detection System.