Long non-coding RNA sequencing (RiboZero) analysis of monocytes-derived dendritic cells

Analysis of lncRNA differential expression in Monocytes-derived Dendritic cells at 18h, Day 3, Day 5, and Day 7. StringTie was used to perform expression level for mRNAs and lncRNAs by calculating FPKM (FPKM=[total_exon_fragments/mapped_reads(millions)×exon_length(kB)]). The differentially expressed mRNAs and lncRNAs were selected with log2 (fold change) >1 or log2 (fold change) <-1 and with parametric F-test comparing nested linear models (p value < 0.05) by R package Ballgown. Freshly prepared buffy coats were collected from healthy donors (Oklahoma Blood Institute, Oklahoma City, OK, USA) and CD14+ monocytes were obtained by density gradient centrifugation and magnetic bead isolation. Monocytes were plated at a density of 2X10^6/ml in DMEM, supplemented with penicillin (100 U/ml), streptomycin (100 mg/ml), and gentamicin (50 mg/ml). After 2 h, the media were substituted with media containing 10% heat-inactivated FBS, rhGM-CSF, and IL-4 (both 50 ng/ml; PeproTech, Rocky Hill, NJ, USA). Media were replaced every 72 hours. Cells were harvested at 18h, day 3, 5, and 7 for total RNA isolation.