GSM3611071: 151007020_5hmC_Seal; Homo sapiens; OTHER
Approximately 2-3 mL of frozen plasma from each subject was processed by centrifuging at 1,350 × g for 12 min twice, then at 13,500 × g for 12 min once, and followed by cfDNA extraction (1-2 ng/sample) using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Germany). Genomic DNA (gDNA) from cfDNA-paired tumor blocks for 7 patients were isolated (30-50 ng/sample) using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Germany) and fragmented by sonication. We constructed 5hmC-Seal libraries according to established protocol.22 DNA samples were first repaired and ligated with adaptors. Next, the T4 bacteriophage enzyme β-glucosyltransferase was used to transfer an engineered glucose moiety containing an azide-group to 5hmC in duplex DNA. A biotin tag was then added to the azide group using Huisgen cycloaddition (“Click”) chemistry. Finally, the 5hmC-containing DNA fragments with biotin tags were captured by avidin beads. The 5hmC-Seal libraries were constructed through PCR amplification and sequenced using the Illumina NextSeq500 platform (PE38) at the University of Chicago Genomics Core Facility. We randomized all samples when preparing the 5hmC-Seal libraries and sequencing. Technical robustness, including reproducibility, of the 5hmC-Seal was demonstrated in our previous report
GEO
GSM3611071
GEO Accession
GSM3611071