treatment: IFN beta, 1000U/ml for 5 days condition: Healthy
Treatment protocol
Human IFNβ (IF014, Merck, KGaA, Darmstadt), IFNα2a (11100-1, PBL assay science, NJ, USA), and IFN IFNα2b (11105-1, PBL assay science, NJ, USA) concentrations were determined through a dose response curve, where expression of ISG15 was used as a marker for activation of the type I IFN pathway (Supplementary figure 1). As described previously (Buang, N. et al., Nature Comms, 1980), IFN α2a, α2b, and β were added at a concentration of 1000U/ml for 5 days before analysis. This concentration of IFNs increased ISG expression to the same degree seen in IFN-High SLE patients and was therefore used in all subsequent experiments. No evidence of cell death was detected after the 5-day period (Supplementary figure 1).
Growth protocol
Peripheral blood mononuclear cells (PBMC) were isolated from the buffy coats of healthy donors, obtained with consent from the Irish Blood Transfusion Services, by density-gradient centrifugation over Lymphoprep (StemCell Technologies). Cells were washed, resuspended at 2.5x106 PBMC/ml in RPMI (Gibco) supplemented with 10% AB human serum (Sigma90 Aldrich), and plated onto non-treated tissue culture plates (Costar). Cells were maintained in humidified incubators for 7 days at 37°C and 5% CO2. Non-adherent cells were removed by washing every 2-3 days. The purities of MDM were assessed by flow cytometry and were routinely > 90% pure.
Extracted molecule
total RNA
Extraction protocol
On day 5 of CT1E, RNA from each treatment group was extracted using the RNeasy® Plus Mini Kit (Cat. No. 74134, Qiagen, Limburg, Netherlands) according to the manufacturer’s instructions. The ‘gDNA eliminator’ column included in this kit removed genomic DNA in all 107 samples. For each experiment, RNA quality and quantity was assessed and only RNA with a RNA integrity Number (RIN) above 9.0 was used for Nanostring analysis. The NanoString nCounter Human Metabolic Pathways Panel (Cat # XT-CSO-HMP1-12, N = 6 per IFN sub110 type), which detects transcripts of 768 metabolic genes.
Label
N/A
Label protocol
N/A
Hybridization protocol
N/A
Scan protocol
N/A
Description
N/A
Data processing
All data were analyzed using nSolver 4.0. Background was subtracted from negative controls and samples were normalized to the positive controls and housekeeping genes. Data were further analyzed by normalized count and fold difference, and differences were compared between the subtypes. Data visualisation was performed in Rstudio (R version 4.3.3) using packages EnhancedVolcano (version 1.10.0), ggplot2 (version 3.4.0), ggrepel(version 0.9.2), and pheatmap (version 1.0.12).
