Epigenetic Biomarker for Osteosarcoma

The investigator want to enroll 100 osteosarcoma participants initially treated in Musculoskeletal Tumor Center of Peking University People's Hospital(PKUPH). All those participants will follow the chemo-protocol for osteosarcoma in PKUPH.8 ml of peripheral blood would be drawed before each cycle of chemotherapy for further analysis. After definitive surgery, participants will need to take 8ml of peripheral blood every 2 months for 6 months. A total of 6 times of blood drawing will need to be done.

In hMe-Seal approach, peripheral blood is collected into EDTA-coated Vacutainers. Plasma is collected from the blood samples after centrifugation at 1 600× g for 10 min at 4 °C and 16 000× g at 10 min at 4 °C. CfDNA is extracted using the Circulating Nucleic Acid Kit(QIAGEN). After that, cfDNA (1-10 ng) is end repaired, 3'-adenylated and ligated to DNA Barcodes using KAPA Hyper Prep Kit (Kapa Biosystems). Ligated DNA is incubated in a solution containing HEPES buffer, UDP-6-N3-Glc and βGT . After that, DBCO-PEG4-biotin is directly added to the reaction mixture. Next, DNA is purified by Micro Bio-Spin 30 Column (Bio-Rad). The purified DNA is incubated with M270 Streptavidin beads (Life Technologies) in specific buffer. The beads are subsequently undergone three 5-min washes each with four kinds of different buffers. All binding and washing are done at room temperature with gentle rotation. Beads are then resuspended in water and amplified with 16 cycles of PCR amplification. The PCR products are purified using AMPure XP beads. Pair-end 38bp sequencing is performed on the NextSeq-500 instrument.