GSM7849754: #1 memory CD8+ T cells, blood, 4 hour stimulation; Homo sapiens; Bisulfite-Seq
PBMCs were isolated from residual blood samples from disposable kits used during routine platelet apheresis via discontinuous gradient centrifugation. CD8+ T cells were pre enriched using human anti-CD8 MicroBeads and the automated magnetic activated cell sorting (autoMACS) system (Miltenyi Biotec) according to the manufacturer's instructions. The cells were stimulated with CMVpp65 overlapping peptide pool (Miltenyi Biotec) at a concentration of 1 µg of each peptide/ml for 4 hours. Subsequently, IFN γ secreting cells were labelled using IFN γ Secretion Assay/Detection Kit (PE) (Miltenyi Biotec; manufacturer's protocol) and antibodies for surface protein detection. Genomic DNA was prepared from flow cytometrically sorted naive CD8+ T cells, interferon-gamma negative memory CD8+ T cells and interferon-gamma positive CD8+ T cells by using DNeasy blood and tissue kit (Qiagen, manufacturer's protocol). Genomic DNA was bisulfite converted using the EZ DNA Methylation Kit (Zymo Research,  manufacturer's protocol) and subjected to library preparation. The bisulfite converted DNA was fragmented by sonication and served as input for library preparation using the Accel-NGS Methyl-Seq DNA Library Kit (Swift Biosciences, manufacturer's protocol).