Spleen dendritic cells from WT mice, positively enriched with a cocktail of anti-CD11c- and PDCA1-conjugated magnetic beads (Miltenyi Biotec), and FACS sorted as CD19(neg), CD3(neg), NK1.1(neg), 120G8(neg/low), CD11c(high), CD11b(positive)
Treatment protocol
Isolated spleen were digested with collagenase (Liberase CI), erythrocytes were lysed with NH(4)Cl, and leukocytes were resuspended in PBS EDTA 5mM before magnetic separation
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from between 7.5x10E5 and 1.5x10E6 cells for each leukocyte subset with the Qiagen micro RNAeasy kit, with on column DNase digestion, according to the manufacturer protocol, yielding between 200 and 700 ng of total RNA for each sample.
Label
biotin
Label protocol
100 ng of RNA from each sample was used to synthesize biotinylated probes, using 2 successive rounds of cRNA amplification with appropriate quality control to ensure full length synthesis according to standard Affymetrix protocols.
Hybridization protocol
Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip MOE-430 2.0 Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400 with the EukGE_WS2v5 protocol.
Scan protocol
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000.
Description
Gene expression data from purified splenic DCs from 10-15 WT C57BL6 mice
Data processing
Raw data were transformed with the Mas5 algorithm, which yields a normalized expression value, and "absent" and "present" calls. Target intensity was set to 100 for all chips.
