GSM1544112: HMEC-hTERT_Bound_MeDP; Homo sapiens; MBD-Seq
Genomic DNA was sheared to a mean fragment length of 300-500 bp through sonication. Methylated-DNA precipitation (MeDP) was carried out using the MethylMiner Methylated DNA Enrichment Kit (Life Technologies) from 1µg of sheared DNA. Pools of 5 independent experiments, library preparation and high throughput sequencing (single-end 50 bp sequencing on Illumina HiSeq 2000) were performed at Beijing Genomics Institute (Hong-Kong, China). MBD2 Chromatin Immunoprecipitations (ChIP) were performed as previously described (Auriol et al. Nucleic Acids Research 2005). Briefly, sheared chromatin (with a mean fragment length between 300 and 500 bp) was obtained by sonication of formaldehyde cross-linked nuclei. ChIP was then performed with a custom-made rabbit polyclonal serum obtained after immunization with peptides corresponding to the N-terminal part of the MBD2 protein (Covalab, Villeurbanne, Lyon) (Perriaud et al. Curr. Pharm. Des 2013) using the ChIP Assay Kit (Merck Millipore, Saint-Quentin-en-Yvelines, France) as specified by the manufacturer’s instruction. Precipitated DNA was finally purified using NucleoSpin Gel and PCR Clean-up kit, (Macherey-Nagel) according to the manufacturer’s protocol “DNA clean-up of samples containing SDS”. DNA-end was repaired to overhang a 3'-dA, then adapters were ligated to the end DNA fragments. DNA fragments with proper size (usually 100-300bp, including adaptor sequence) were selected after PCR amplification.