GSM7697709: ZK_P_mC_3; Mus musculus; MeDIP-Seq
DNA was extracted by adding equal volume of buffered phenol:chloroform:isoamyl alcohol (25:24:1 ratio) and centrifuged in Phase-Lock tubes at 15,000 RPM at room temperature. Supernatant was transferred to clean tubes. An equal volume of isopropanol was then added to the supernatant and mixed well at room temperature to precipitate the DNA. The DNA was then centrifuged at 10,000g for 10 minutes at room temperature, and then washed in 70% ethanol. After all ethanol was removed, the DNA pellet was eluted in nuclease-free water and incubated at 55 for 1 hour before storing at -20. 3mg of genomic DNA was sonicated to 300-400 base pairs using a Covaris focused ultrasonicator. The DNA fragments were then subjected to end repair, A-tailing, adaptor ligation and USER digestion using the NEBNext Ultra II DNA Library Prep kit for Illumina (New England BioLabs, E7645S) according to the manufacturer's protocol. Following USER digestion and purification, DNA was denatured for 10 minutes at 95°C and immunoprecipitated overnight at 4°C with 4mL of either 5mC antibody (Active Motif, 39649) or IgG antibody (Sigma 12-371) in IP buffer (500mM Tris-HCl, pH 7.4, 750mM NaCl and 0.25% TritonX). The mixture was then incubated with Protein G coated Dynabeads for at least 2 hours at 4°C, washed with ice cold IP buffer and finally washed in ice cold high salt (300mM NaCl) IP buffer. After the final washing, the beads were treated with 200mL digest buffer (1X TE Buffer, pH 7.4, 0.25% SDS, 0.25% Proteinase K (2.5mg/mL)) and shaken at 1000rpm for 2 hours at 55°C. The methylated DNA was recovered by phenol:chloform:isoamyl alcohol (25:24:1) extraction followed by precipitation in 3X volume of 100% ethanol supplemented with 3mL glycogen(5mg/mL) and 15mL NaAC pH 5.2 overnight at -20°C. The next day, the DNA was pelleted and washed with 75% ethanol and dissolved in nuclease-free water. Illumina indexes were added, PCR enriched and purified using the NEBNext Ultra II DNA Library Prep kit for Illumina following the manufacturer's protocol.