GSM8218866: Oc4 CD14+ Monocytes of HGSOC patients; Homo sapiens; Bisulfite-Seq
The peripheral blood mononuclear cells (PBMCs) were separated from whole blood by density gradient centrifugation using Lymphosep, Lymphocyte Separation Media (#L0560-500, Biowest, France), 1.077 g/ml density, at 600g for 30 minutes. The isolation of monocytes from PBMCs was performed using positive magnetic selection with CD14+ MACS beads (#130-050-201, Miltenyi Biotech, Germany), resulting in 90–98% monocyte purity as confirmed by flow cytometry. After monocyte isolation, the samples were washed twice with DPBS without calcium and magnesium at 300 g for 10 minutes. Subsequently, the cell precipitate was lysed using lysis buffer RLT (#79216, Qiagen, USA) and stored at -80°C until further experiments. Lysed monocytes were used for DNA isolation and reduced representation bisulfite sequencing (RRBS-Seq). DNA were extracted from lysed monocyte samples using AllPrep DNA/RNA/miRNA Universal Kit (#80224, Qiagen, USA). The quality of DNA was assessed by TapeStation 4150 automated electrophoresis system (#RRID: SCR_019393, Agilent Technology, USA). The quantity of DNA was assessed by Qubit 4 fluorometer (#RRID: SCR_018095, ThermoFisher Scientific, USA). Samples were purified using magnetic beads AMPure XP (Beckman Coulter) and concentration was controlled using Fluoroskan. Libraries were prepared using Zymo-Seq RRBS Library Kit (#D5461) and Zymo-Seq UDI Primer Plate (#D3096) (Zymo Research, USA), according to the manufacturer's instructions. Sequencing was performed by the Illumina NovaSeq 6000 platform (Illumina, USA).