Nanostring Immune Profiling Protocol

In cohort 1, RNA was purified using AllPrep^®DNA/RNA/miRNA Universal Kit according to manufacturer’s instructions (Qiagen). For multiplex gene expression analysis, the Nanostring nCounter^® platform was used. 100 ng total RNA in 7-20 µl (5-14ng/µl) was used as input. Samples were hybridized to target specific barcodes consisting of a capture probe and a reporter probe using the Nanostring nCounter^® PanCancer Immune Profiling panel (NanoString Technologies Inc, Seattle, WA, USA). After hybridization, target RNA was isolated by magnetic bead sorting and immobilized by applying an electric current as described in the hybridization protocol in the nCounter XT Assay user manual (NanoString Technologies Inc). Gene expression data were normalized with CD8A and CD8B that are stably expressed in CD8⁺ T cells (34 (link)). Two outliers were excluded after nSolver gene expression analysis, why only 38 genotype-selected HS were analysed in cohort 1.