GSM9157568: ME/CFS RRBS, patient #007 at 24hr; Homo sapiens; Bisulfite-Seq
Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by layering it onto Ficoll-Paque, followed by centrifuging at 400×g to separate plasma from PBMCs and the PBMCs from other cells. The removed PBMC layer was diluted with PBS and then pelleted at 100×g. The pellet was washed in PBS and stored at −80°C in a solution of FCS containing 10% DMSO. DNA was extracted from 200 μL of the PBMC fraction using the Illustra Blood Genomic Prep Mini Spin Kit, following the manufacturer's instructions. Elution was performed using the kit's EB buffer, and DNA concentration was measured with a Qubit 2.0 fluorometer according to the Qubit dsDNA HS Assay Kit protocol. 500 µg of genomic DNA was digested using 160 U of the MspI restriction enzyme. After end repair and adenylation of the 3′ ends, adaptors were ligated to the DNA fragments. Bisulfite conversion was carried out using the EZ DNA Methylation Kit (Zymo Research Corp, USA) according to the manufacturer's instructions. A semi-quantitative PCR was performed on the bisulfite-converted DNA to determine the optimal number of amplifi-cation cycles required for the final large-scale PCR of the complete library. Following PCR amplification, DNA was size-selected using magnetic beads (AMPure XP beads from the TruSEq DNA nanokit -Beckman Coulter USA), isolating fragments between 40–220 bp to construct the RRBS libraries while minimizing adaptor contamination. The purified DNA was assessed for quality using a BioAnalyzer (Agilent, USA) and Qubit (Thermofisher Scientific, USA) measurements, followed by further purification with AMPure XP Beads (Beckman Coulter, USA).