Peripheral blood mononuclear cell (PBMC) preparations and HLA Genotyping

PBMCs were isolated from whole blood using Ficoll-Paque density gradient centrifugation (GE Healthcare, Chicago, IL). Whole blood was diluted to 50 mL with 1x PBS + 2% FBS (fluorescence activated cell sorting (FACS) buffer), layered over Ficoll-Paque and centrifuged at 400 x g for 30 min with no brakes. The buffy coat layer was then moved to a new tube and washed twice with FACS buffer.

DNA was extracted from the cells using a DNeasy Blood and Tissue kit (Qiagen, Maryland, USA) following the kit instructions. The HLA typing was performed by Sirona Genomics (Mountain View, CA), an Immucor Company. The assay, based on a previous publication (Wang et al., 2012), was performed using the MIA FORA NGS HLA typing assay for the class I loci. The full-length amplicons for the class I loci were amplified and pooled. These samples were then fragmented, and tagged with unique index adaptors. The samples were pooled and sequenced on the Illumina MiSeq, and the HLA type was determined using the MIA FORA NGS HLA typing software. The Sirona Genomic HLA typing method has been validated by the Histocompatibility, Immunogenetics and Disease Profiling Laboratory of the Stanford University School of Medicine using 50 reference cell lines.