Computational protocol: Human CD141+ Dendritic Cell and CD1c+ Dendritic Cell Undergo Concordant Early Genetic Programming after Activation in Humanized Mice In Vivo

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Protocol publication

[…] Total RNA from purified DC subsets was prepared by resuspending between 1 × 105 and 1 × 106 cells in TRIzol Reagent (Life Technologies) followed by chloroform extraction and isopropanol precipitation. RNA quantity and integrity was measured using the Bioanalyzer RNA Nano chip (Agilent Technologies), with all RNA integrity numbers ranging between 8.4 and 9.7. cDNA was generated from 450 ng RNA and converted to cRNA using an Illumina TotalPrep RNA Amplification Kit (Ambion). A 14 h in vitro transcription was performed. Amplified cRNA (750 ng) for each sample was hybridized onto HumanHT-12 v4 Expression BeadChip Kit (Illumina) according to the manufacturer’s protocol and scanned on an Illumina BeadArray Reader (Illumina). Quality control, normalization, and log 2 transformation of the raw expression data were performed using the Lumi Bioconductor package () and integrated into the Stemformatics platform www.stemformatics.org () for visualization. Transcripts differentially expressed by CD141+ DC and CD1c+ DC were identified using ANOVA analysis with p value cut-off threshold of Bonferroni correction p < 0.01 and log(2) 3-fold difference. The data have been deposited in the GEO database, accession # GSE99666, and on Stemformatics http://www.stemformatics.org/datasets/search?ds_id=6612. Publically available data sets and gene signatures for human DC subsets GSE35457 () were processed similarly for comparison. […]

Pipeline specifications

Software tools Beadarray, lumi
Databases Stemformatics
Application Gene expression microarray analysis
Organisms Homo sapiens, Mus musculus