GSM2299499: MBD_Ctrl_Chow_rep_4; Mus musculus; MBD-Seq
Genomic DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen), and quantified using Qubit dsDNA HS kit (Life Technologies). Purified DNA was sonicated to achieve fragments between 100-1000 bp in size. Methylated DNA was captured using MethylMiner (Invitrogen). Methylated DNA was captured by beads conjugated with methyl-binding-domains (MBD). Captured DNA was eluted using a high-salt buffer, followed by ethanol precipitation. Recovered DNA was prepared for sequencing by adenylation using dATP and Klenow DNA polymerase (3’ to 5’ exo minus) (New England Biolabs) followed by ligation to TruSeq adapters (Illumina). Ligation products were purified using AMPure Beads, subjected to an ethanol-based cleanup and amplified by PCR (30s 98°C; (10s 98°C, 30s 60°C; 30s 72°C) x 18-25 cycles; 5 min 72°C) using Phusion DNA Polymerase (New England Biolabs). PCR products were purified using AMPure Beads (Beckman Coulter). Libraries were quality-controlled using a Bioanalyzer instrument (Agilent)