Supporting Information

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Table 1. Selected genes regulated upon pDC activation

Changes in gene expression were expressed as the fold difference of treated cells compared with untreated pDCs after 18 h of culture.

SI Materials and Methods

After incubation in culture medium alone or supplemented with p17 or CpG, total RNA was extracted from pDC (2.5 ´ 10⁵ cells per sample) using the Qiagen RNA extraction easy kit. The quality and integrity of the isolated total RNA were controlled by running samples on an Agilent Technologies 2100 Bioanalyzer (Agilent Technologies). For biotin-labeled target synthesis, reactions were performed using protocols supplied by the manufacturer (Affymetrix). In the first of two rounds of RNA amplification, nonlabeled cRNA was prepared using Promega P1300 RiboMax Kit (Promega) for T7 amplification.

For the second round of amplification, the precipitated and cleaned cRNA was converted into cDNA using random hexamers (Amersham Biosciences). Second-strand synthesis and probe amplification were carried out as in the first round, apart from an incubation with RNase H before first-strand synthesis to digest the cRNA and the use of the T7T23V oligo to initiate the synthesis of the second strand. The concentration of biotin-labeled cRNA was determined by UV absorbance. In all cases, 12.5 mg of each biotinylated cRNA preparation were fragmented and placed in a hybridization mixture containing four biotinylated hybridization controls (BioB, BioC, BioD, and Cre). Samples were hybridized to an identical lot of Affymetrix HG_U133 plus 2.0 GeneChips, according to the manufacturer's protocol (Affymetrix). After hybridization, the GeneChips were washed, stained with SA-PE, and read using an Affymetrix GeneChip fluidic station and scanner. Scanned images and raw data were processed using Robust Multiarray Average as described in SI Ref. 1.