GSM7703418: HS_5; Homo sapiens; Bisulfite-Seq
A total of 15 mL of peripheral venous blood was collected in EDTA vacuettes through venipuncture from AF patients (at hospitalization time and before any treatment administration) and from HS. Blood samples were centrifuged at 1900 x g 10 min at + 4°C within 1 hour and aliquots of plasma were stored at -80°C for long-term storage. Peripheral blood mononuclear cells (PBMCs) were isolated via density centrifugation using Ficoll-Paque™ PLUS (Cytiva) according to the manufacturer's protocol. CD4+ T cells were purified starting from 5x107/mL of  PBMCs by negative immunomagnetic selection using EasySep™ Human CD4+ T Cell Isolation Kit (STEMCELL Technologies) as previously described. To perform genome-wide DNA methylation analysis, genomic DNA from 10 AF patients and 11 HS (set population) were analyzed by RRBS at the Genomix4Life S.r.l. (Baronissi, Salerno, Italy). DNA concentrations for each sample was assayed with Qubit fluorometer (Thermo Fisher Scientific) and its quality assessed with the TapeStation 4200 system (Agilent Technologies, Inc.). Indexed libraries were prepared starting from 100 ng/ea purified DNA with Ovation RRBS Methyl-Seq System (Tecan Group Ltd.) according to the manufacturer's instructions. Libraries were quantified using the TapeStation 4200 system (Agilent Technologies, Inc.) and Qubit fluorometer (Thermo Fisher Scientific), then pooled such that each index-tagged sample was present in equimolar amounts. The pooled samples were subject to cluster generation and sequencing using an Illumina NextSeq 550Dx (Illumina, Inc.) in a 2x76 paired-end format.