DCs were stimulated with: pRNA (15μg/ ml) and Hiltonol (10 μg/ml) for 16 hours.
Growth protocol
Cells were obtained from aphaeresis of 5 different donors. Peripheral blood mononuclear cells (PBMCs) were isolated by using Ficoll density centrifugation (Lymphoprep; Axis-Shield PoC AS, Oslo, Anti-Human Lineage Cocktail 1 in FITC (LIN1) (BD Bioscience Pharmingen, San Jose, CA) containing antibodies for CD3, CD14, CD16, CD19, CD20, CD56 receptors, together with the anti-FITC conjugated magnetic microbeads of Miltenyi Biotec (Bergisch-Gladbach, Germany) were used to deplete the LIN1+ cell fraction, by following manufacturer’s instructions. Next, CDC1were further purified by sorting (flowcytometry) using anti–BDCA-3–APC combined with anti-HLA-DR-PE-Vio770 (Miltenyi Biotec) to a purity of 99.9%. DCs were cultured in X-VIVO-15 medium (Lonza, Basel, Switzerland) supplemented with 2% human serum (Sanquin).
Extracted molecule
total RNA
Extraction protocol
RNA was harvested using Trizol (Invitrogen, MA, USA), following the standard protocol. Quality control of the isolated RNA (concentration, RIN, 28S/18S and size) was performed with Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA).
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol by ThermoFisher (Clariom D Expression Analysis Technical Manual revision B.0, ThermoFisher).
Hybridization protocol
Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Drosophila Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
Scan protocol
According to the standard Affymetrix protocol by ThermoFisher (Clariom D Expression Analysis Technical Manual revision B.0, ThermoFisher).
Data processing
Expression intensities from CEL files were converted to an expression matrix using the bioconductor packages affycoretools, version 1.70.0, and oligo, version 1.62.2.
