GSM9520884: 16106 - EM-Seq; Homo sapiens; OTHER
Whole blood DNA was extracted from PAXgene blood RNA tubes. Briefly, frozen tubes were thawed at room temperature and centrifuged according to the manufacturer's protocol. The resulting pellet was then washed with nuclease free water, resuspended in sterile PBS, and split in half. DNA was extracted from one half of the suspension using the Mag-Bind Blood & Tissue DNA HDQ Kit (Omega Bio-Tek) on the Kingfisher Flex (Thermo) instrument. The other half of the suspension was used for RNA extraction. The concentration and integrity for DNA was evaluated by Picogreen Assay and Agarose Gel electrophoresis. Whole genome methylation libraries were constructed using 200ng gDNA input with NEBNext® Enzymatic Methyl-seq Kit as per manufacturer instruction with minor modifications for automation. Library concentration was measured using the Quant-iT™ PicoGreen® dsDNA Reagent (Thermo), and library size distribution and quality were checked using a DNA chip on Caliper Gx Touch (Revity). Library quantification was further refined by qPCR using the KAPA Library Quantification Kit (Roche). Paired-end sequencing (2 × 150 bp, approximately 30X coverage per sample) was carried out on an Illumina NovaSeq 6000 Plus platform (Illumina) following the manufacturer's recommended protocols.