GSM5628993: 5hmC_Seal_E61; Homo sapiens; OTHER
CfDNA samples from T2D patients were prepared from peripheral blood with EDTA anticoagulation. Briefly, 5 mL of peripheral blood was collected from each subject, and plasma was separated within 4 h by centrifuging twice, one at 1 350× g for 12 min, and another at 13 500× g for 5 min. The processed plasma samples were stored at –80 °C immediately. CfDNA was extracted using the Circulating Nucleic Acid Kit (Qiagen, Germany) according to the manufacturer's instructions. On average, 1-2 ng of cfDNA was obtained from each cfDNA sample. The quality of cfDNA was checked using the standard molecular biology approach Each cfDNA sample was first repaired and ligated with adaptors. Next, the T4 bacteriophage enzyme β-glucosyltransferase (β-GT) was used to transfer an engineered glucose moiety containing an azide-group to 5hmC in duplex DNA. A biotin tag was then installed onto the azide group using Huisgen cycloaddition (click) chemistry, followed by capturing of 5hmC-containing DNA fragments using avidin beads. The 5hmC-Seal library for each cfDNA sample was then constructed through PCR amplification, followed by paired-end sequencing (PE38) at Shanghai Epican Genetech, Co. Ltd. (Shanghai, China), using the Illumina NextSeq500 platform according to Illumina's protocol. We randomized cfDNA samples when constructing the 5hmC-Seal libraries and sequencing.