title: Illumina HiSeq 2000 paired end sequencing; Genome-wide plasma cell-free DNA methylation profiling in pancreatic cancer patients using MeDIP-seq
design: Genome-wide plasma cell-free DNA methylation profiling in pancreatic cancer patients using MeDIP-seq
library_construction_protocol: All pancreatic cancer blood samples were obtained from patients with ductal adenocarcinoma and control blood samples were from healthy volunteers. 5 ml peripheral blood was collected using EDTA anticoagulant tubes. The blood specimens were collected before surgery and drug treatment. The plasma was purified by centrifuged for 15min at 1500g within 6 h. The plasma was stored at -80℃ until DNA extraction. cfDNA were extracted from 800-μl aliquots of plasma using QIAamp Circulating Nucleic Acid Kit (Qiagen, 55114) according to manufactory protocol and quantified with Bioanalyzer 2100 (Agilent Technologies).  The cfDNA MeDIP-seq library was prepared as what we described previously (W. Xu et al., 2019). Basically, about 20ng cfDNA was ligated with Illumina barcode adapters using KAPA Hyper Prep Kit (KAPA, KK8502). The constructed cfDNA libraries were denatured at 95oC, 10min. The methylated cfDNA was separated from the cfDNA libraries by immunoprecipitation using 5-Methylcytosine (5mC) Monoclonal Antibody (Epigentek, A-1014). MeDIP DNA was further amplified using Q5 High-Fidelity DNA Polymerase (NEB, M0491).
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: anti-5-Methylcytosine (EpiGentek, 16-663)