GSM6330094: HEK293T, MeDIP, EV; Homo sapiens; MeDIP-Seq
Genomic DNA was isolated using QIAamp DNA Mini Kit (Qiagen). For each MeDIP experiment, 2 µg of DNA was fragmented into ~100-500 bp by sonication. DNA fragments were subjected to end repair and adapter ligation using End Repair/dA-Tailing Module (NEB, E7442) and Ligation Module (NEB, E7445), respectively. Immunoprecipitation was performed with 2 µg of anti-5mC (Abcam, ab214727) in IP buffer (﻿100 mM sodium phosphate, pH 7.0, 1.4 M NaCl and 0.5% Triton X-100). After overnight incubation at 4°C, 20 µl pre-washed protein A/G magnetic beads (Thermo Scientific) was added to the IP buffer, followed by an additional incubation at 4°C for 2 h. Beads were washed with IP buffer, and DNA was purified with ﻿phenol:chlorophorm:isoamyl alcohol after proteinase K digestion. The isolated DNA was amplified by PCR and size selected for deep sequencing