title: HiSeq X Ten paired end sequencing; Genome-wide plasma cell-free DNA methylation profiling in lung cancer patients using MeDIP-seq
design: Genome-wide plasma cell-free DNA methylation profiling in lung cancer patients using MeDIP-seq
library_construction_protocol: All lung cancer blood samples were obtained from patients with adenocarcinoma and control blood samples were from healthy volunteers. All blood from lung cancer patients and controls (∼5 ml), was collected in tubes containing EDTA as anti-coagulant. Centrifuge blood samples for 15 min at 1500 x g, and then transfer plasma to clean tubes. The plasma was aliquoted in ~1 ml fractions to avoid multiple freeze-thaw times and stored at -80℃ until DNA extraction. The cfDNA was extracted from 1ml plasma using QIAamp Circulating Nucleic Acid kit (Qiagen, 55114) according to the manufacturer's protocol. Qubit (Invitrogen) was used to analysis the concentration of cfDNA in the plasma. The distribution of cfDNA size was estimated by the Agilent Bioanalyzer 2100. The obtained cfDNA was performed for end repair, dA overhanging, Illumina sequencing adapter ligation to get original library. Then, the library was denatured at 95°C for 10 min, immediately ten minutes on ice, then incubated with 5-Methylcytosine (5-mC) Monoclonal Antibody (Epigentek, A-1014) that was bound on Magna ChIP Protein A+G Magnetic beads (Millipore, 16-663) for 4°C overnight, and decrosslinked subsequently. The products were amplified using Q5 High-Fidelity DNA Polymerase (NEB, M0491). The PCR reactions were then purified using AMPure XP beads (Beckman). Amplificated libraries were detected DNA fragmentation size by Agilent Bioanalyzer 2100 (Agilent Technologies) and determined concentration with the Qubit assay (Invitrogen).
attributes.tag: Experimental Factor: individual
attributes.value: patient3
attributes.tag: Experimental Factor: disease
attributes.value: lung adenocarcinoma
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: anti-5-Methylcytosine (EpiGentek)