Agilent s 2100 bioanalyzer system

Total RNA was extracted from WT and DOXR cells using the RNeasy Mini kit (Qiagen). RNA quality was confirmed using Agilent's 2100 Bioanalyzer system (Agilent Technologies, USA). RNA sequencing was performed using the Illumina NextSeq 500 sequencing platform (Illumina, USA). Biological functions were determined using the ingenuity pathways analysis (IPA) web-based bioinformatics software (Qiagen). A two-fold change in DOXR gene expression was used as the cut-off value for genes with a significant change in expression compared to the WT cells.

Human pDCs were isolated as described above and purity was checked by flow cytometry (>90%). PDCs (approx. 100,000 cells, n = 3) were challenged overnight with CpG-A (2.5–5 µM) in the absence or presence of oxLDL, after which the cells were washed and suspended in DEPC RNase-free water. Unstimulated pDCs were used as controls. Total RNA was extracted from the cell suspensions using the RNeasy Micro kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol for extraction of RNA from microdissected cryosections. DNA was removed by using 50U DNase I (Qiagen). RNA quantity and quality were determined using the NanoDrop-1000 Spectrophotometer and the Agilent’s 2100 Bioanalyzer System (Wilmington, DE, USA; Agilent Technologies, Palo Alto, CA, USA); average yield was 115 ng RNA; RNA integrity numbers were >6.5.