title: Acetyl_rep1
organism: Homo sapiens
description: Protocols: Monocytes were prepared from peripheral blood of healthy individuals using the method of plastic adhesion. All volunteers provided the written informed consent to participate in the study. Volunteers were included in the study if they had no manifestations of atherosclerosis. All experiments were repeated on monocytes/macrophages isolated from 3 different donors. The isolated cells were incubated at 37C in cell culture incubator under 5% CO2 for 2h and rinsed three times with RPMI-1640 culture medium to remove non-adherent cells. The adherent cells were detached mechanically and plated in culture plates (Corning). Cells were cultured in RPMI-1640 medium supplemented with 10% of human serum, 50 ng/mL human M-CSF (PeproTech), and 25 ng/mL IL-10 (PeproTech). Medium was refreshed on day 3 and replaced with serum-free X-VIVO medium (Lonza Group Ltd.) on day 6. The obtained monocyte-derived macrophages were used for experiments on day 7. On day 7, X-VIVO medium was refreshed, and 50 µg/mL of LDL normalized by total protein measured using Lowry method) or 0.4 µL/mL of latex beads suspension in X-VIVO medium were added. Cells were incubated for 24 h. Total RNA was isolated from cells and purified using NucleoSpin RNA and RNase-free DNase digestion, according to the manufacturer's instructions (MACHEREY-NAGEL). RNA-seq libraries were prepared from poly(A)-enriched mRNA using NEBNext Ultra RNA Library Prep kit for Illumina, according to the manufacturer's protocol (New England BioLab).