Ncounter human immunology v2 panel

RNA from whole blood was isolated from Tempus blood RNA tubes according to the manufacturer’s protocol (Tempus Spin RNA Isolation Kit, Thermo Fisher Scientific). Moreover, 50 ng of RNA was hybridized to reporter and capture probe sets of the nCounter Human Immunology v2 panel (Nanostring Technologies) at 65 °C for 24 hours. For in vivo experiments, 100 μL of whole blood collected 1 day postvaccination was lysed with BD PharmLyse reagent. RNA was subsequently extracted with the QIAGEN RNAeasy micro kit. 50ng of RNA was hybridized to the nCounter Mouse Inflammation v2 panel (NanoString Technologies) as previously described [28 (link)]. Unsupervised PCA performed to visualize variability between immunization routes was performed with Partek Genomics Suite Analysis v.7 software. Hierarchical clustering was performed with Seaborn’s Clustermap function in Python version 3.

We harvested CFPAC-1 tumors from humanized mice 31 days post injection of CAdTrio. Total RNA was extracted from whole tumors using the RNeasy Plus Mini kit and quantified using the NanoDrop 2000 (Thermo Fisher Scientific Inc.). The Baylor Genomic & RNA Profiling Core performed RNA expression profiling with the nCounter Human Immunology V2 Panel (NanoString Technologies). Data quality control, normalization, and advanced analysis were performed using nSolver 4.0 analysis software following NanoString analysis guidelines. Supplementary gene expression tables show expression level >2 and p < 0.05.

The mRNA expression of 600 targets was analyzed with the nCounter Human Immunology V2 panel including 20 customized probes (Nanostring Technologies) on the nCounter platform (Nanostring Technologies) using 100 ng of RNA per skin sample. This commercial panel was extensively validated in-house for accuracy, repeatability, and reproducibility before analyzing the study samples. A quality check was run for each sample before including it in the analysis. Data were normalized and analyzed using ROSALIND (ROSALIND, Inc.). Basically, housekeeping probes to be used for normalization are selected based on the geNorm algorithm as implemented in the NormqPCR R library. Clustering of genes for the final heatmap of differentially expressed genes was done using the Partitioning Around Medoids method using the fpc R library that takes into consideration the direction and type of all signals on a pathway, the position, role, and type of every gene. The z-values of each gene were then calculated for the selected patients to generate heatmaps and determine specific classifiers. The autoinflammation gene signature score was calculated as the sum of z-values of IL1A, IL1B, CXCL1, and CXCL8 for each patient.

For each sample, 100 ng of total RNA from keratinocytes was hybridized for 19.5 hours with probes from the nCounter Human Immunology V2 Panel containing 579 target genes and 15 reference genes (NanoString Technologies, Seattle, WA, USA). The samples were examined using nCounter SPRINT profiler (NanoString Technologies) according to the manufacturer’s instructions. Before the differential expression analyses, the raw data were normalized using nSOLVER 4.0 (NanoString Technologies) by performing background subtraction, positive control normalization and normalization to housekeeping genes (EEF1G, GAPDH, GUSB, OAZ1, and RPL19). Genes were filtered to only include genes with an average normalized count ≥ 20 corresponding to 271 genes. Differential gene expression analysis was performed by multiple paired t-tests with a false discovery rate < 0.05 using the two-stage step-up method of Benjamini, Krieger and Yekutiel in GraphPad Prism 9.0 software (31 (link)).

NanoString profiling was performed using the nCounter Human Immunology v2 Panel (NanoString Technologies, Inc. Cat# XT-CSO-HIM2-12). 50 ng of RNA was mixed with 8 ul of reporter probeset and 2ul of capture probeset, and incubated at 65 °C for 24 h. Hybridised samples were loaded onto an nCounter Sprint cartridge and imaged on a nCounter Sprint system. Raw and normalised counts were derived with the NanoString nSolver 4.0 software as per manufacturer’s protocols. nCounter probe sets included negative and positive controls that were used for background thresholding, and normalizing samples for differences in sample input or hybridisation.

For gene expression analysis, total RNA was purified from two T25 flasks containing monolayers of uninfected and infected AV3 cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA quality was assessed by spectrophotometry (Trinean, Gentbrugge, Belgium). A gene expression assay was carried out as previously reported [27 (link)] using the nCounter Human Immunology v. 2 Panel (nanoString Technologies, Seattle, WA, USA) that allows analysis of 580 immune-related genes. Duplicate samples have been used; experiments have been repeated three times.