Transcriptome profiling of peripheral blood mononuclear cell subsets

Transcriptome profiling was conducted on 2 replicate samples of total peripheral blood mononuclear cells (PBMC) and isolated PBMC subsets. Gene expression profiling was conducted on total PBMC and parallel isolated subsets of CD+/CD4+ T lymphocytes, CD3+/CD8+ T lymphocytes, CD19+ B lymphocytes, CD3-/CD14-/CD56+ NK cells, CD66b+ neutrophils, CD14+ monocytes, CD14+/CD16- classical monocytes, CD14+/CD16+ non-classical monocytes, CD1c+(BDCA1+)/CD19- myeloid dendritic cells (DC1), CD303+(BDCA2+) plasmacytoid dendritic cells (DC2), and CD141+(BDCA3+)/CD14- myeloid dendritic cells (DC3). Samples of each cell population were isolated from PBMC (derived from ficol gradient centrifugation of buffy coat blood samples). B, T, and NK cells were isolated from total PBMC by flow cytometry. Neutrophils, monocytes, and DCs were isolated by flow cytometric sorting of PBMC that were pre-depleted of CD3+ and CD19+ cells by immunomagnetic positive selection (MACS microbeads; Miltenyi Biotech). Flow cytometry indicated > 99% purity of all cell populations. Two samples of each cell type were pooled to create a total of two independent replicate assay samples for RNA profiling. One replicate set of neutrophil samples yielded sub-optimal RNA and failed post-assay validity checks; that sample has been deleted from the data, so there is only 1 replicate samples of neutrophil data.