Total RNA, including small non-coding RNAs, was extracted using the mirVana RNA isolation kit (Ambion, Inc, Austin, TX, USA) according to manufacturer’s protocol. RNA was quantified using a ND-1000 spectrophotometer (Nanodrop Technology®, Cambridge, UK) and RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent technologies, Santa Clara, CA, USA). The 260/280 ratio were between 1.7 and 2.1 and the RINs (RNA Integrity Numbers) greater than 6 for all samples.
Label
Biotin
Label protocol
Total RNA (100ng) was transcribed and labeled into amplified RNA (aRNA) using the 3’IVT express kit (Affymetrix Inc., Santa Clara, CA) according to manufacturer’s instruction on a GCAS (Gene Chip Array Station).
Hybridization protocol
Fragmented and labeled aRNA was hybridized to the Affymetrix U133 Plus 2.0 chips according to manufacturer’s instructions. Wash and stain protocol FS450_0004.
Scan protocol
GeneChips were scanned using the GC 30007G scanner.
Description
CD123+ plasmacytoid dendritic cells (pDCs), CD11c+ myeloid dendritic cells (mDCs) and CD14+ monocytes were isolated using the EasySep® Human pDCs Enrichment Kit, a custom EasySep® Human mDCs Enrichment Kit (containing antibodies directed against CD3, CD19, CD14, CD16, CD19, CD25, CD34, CD56, CD66b, GlyA, CD85g, CD45RA, CD123 and dextran) and the Human CD14 positive Selection Kit (all from Stemcell Biotechnologies) respectively according to manufacturer’s instructions.
Data processing
Data was normalized in R/Bioconductor using the robust multi-array average (RMA) expression measure. Probesets were annotated using the latest annotation available in Bioconductor and curated using an internal Roche probe inspector tool, which updates gene annotation per probeset based on the latest human genome draft release. As an additional quality measure, probesets were also curated using the tool for sequence specificity; probesets mapping to non-unique locations in the genome were flagged and ignored in subsequent analysis. A total of 20,367 out of 54,675 probesets passed the QC and curation processes, corresponding to 11,629 unique genes.
