Illumina HiSeq 2000 paired end sequencing; RNA-seq of primary human monocytes (4 healthy donors)
RNA-seq of primary human monocytes (4 healthy donors)
Peripheral blood mononucleated cells (PBMC) were obtained from healthy donor buffy coats (Etablissement Français du sang, Rungis, France), separated on Pancoll (Pan-Biotech, Dutscher, Brumath, France). Peripheral blood monocytes were enriched using the pan monocyte isolation kit (Miltenyi Biotech, Paris, France) and the AutoMacs system. Monocyte subsets were labeled with CD14-PE and CD16-APC and sorted by flow cytometry (Influx, BD biosciences). Total RNA was isolated from primary monocytes or macrophages with RNA/DNA/Protein Purification plus Norgen kit. The RNA integrity (RNA Integrity Score ≥ 7.0) was checked on the Agilent 2100 Bioanalyzer (Agilent) and quantity was determined using Qubit (Invitrogen). SureSelect Automated Strand Specific RNA Library Preparation Kit was used according to manufacturer's instructions with the Bravo Platform. Briefly, 100 ng of total RNA sample was used for poly-A mRNA selection using oligo(dT) beads and subjected to thermal mRNA fragmentation. The fragmented mRNA samples were subjected to cDNA synthesis and were further converted into double stranded DNA using the reagents supplied in the kit, and the resulting dsDNA was used for library preparation. The final libraries were processed using Illumina TruSeq Stranded mRNA Library Prep Kit.
Experimental Factor: cell type
CD14-low, CD16-positive monocyte