GSM7820724: DCreg 1011; Homo sapiens; Bisulfite-Seq
Leukapheresis products were obtained under an IRB-approved protocol (Institute for Transfusion Medicine, Pittsburgh, PA). Monocyte-enriched, elutriated fractions recovered from 6 normal human donor leukapheresis products were used for generation of DCregs and monophosphoryl lipid A (MPLA; TLR4 ligand low toxicity derivative of bacterial lipopolysaccharide (LPS))-matured sDC. In instances where the elutriated monocyte fraction purity was <90%, CD14-specific immunobeads (Miltenyi, San Diego, CA) were used according to the manufacturer's protocol to increase monocyte purity to >90%. After 7 days culture, monocyte-derived DCreg and sDC were harvested. The viability of DCregs and sDCs, determined by trypan blue staining, was routinely > 95%. A portion of each DC group was snap-frozen in liquid nitrogen and stored at -80°C for Whole Genome Bisulfate Sequencing (WGBS) DNA Methylation analysis. Portions of the remaining DC were either antibody-stained for key surface molecules, HLA-DR, CD11c, T cell costimulatory and coinhibitory molecules and CD163 (clone RM3/1, Biolegend) and flow cytometry data analyzed using Flow Jo software. DNA was extracted from frozen cell pellets and underwent bisulfite treatment. The bisulfite modified DNA-sequencing library was generated using the Accel-NGS Methyl-Seq DNA library kit (Swift Biosciences) per the manufacturer's instructions.