GSM7594057: 4B-53; Homo sapiens; OTHER
Plasma was separated from whole blood by centrifuge 1350 g for 10 minutes at 4 °C. Plasma cfDNA was extracted using QIAamp Circulating Nucleic Acid Kit (QIAGEN, Germantown, MD) following manufacturer's instructions. Briefly, cfDNA was ligated with barcode adaptors after end repair and A-tailing. Then cfDNA was purified and was incubated with 50 mM HEPES buffer (pH 8.0), 25 mM MgCl2, 100 μM N3-UDP-azide-glucose and 1 μM T4 Phage β-gulcosyltransferase at 37 °C for 1 hour. After purification, cfDNA was incubated with 1 μL DBCO-PEG4-DBCO (a click chemistry tool, 4.5 mM stock in DMSO) at 37 °C for 2 hours. Next, the cfDNA was purified and incubated with 5 μL pre-washed C1 Streptavidin beads (Thermo Fisher Scientific, Waltham, MA) for 15 min. The beads were subsequently washed for 5 minutes eight times. The captured DNA fragments were amplified with 15-18 cycles of PCR. The PCR products were purified with AMPure XP beads according to the manufacturer's instructions. The concentration of DNA library was measured using a Qubit Fluorometer with dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA) and Bioanalyzer 2100 with Agilent High Sensitivity Kit (Agilent Technologies, Santa Clara, CA). nano-hmC-Seal