GSM7033617: A5_equal_IP; Homo sapiens; MeDIP-Seq
Frozen PBMCs were centrifuged twice at 600x g for 10 minutes at 20°C and washed with RPMI+ 20% FBS. The cell pellet was resuspended in PBS to achieve a final volume of 7,500 cells per 1µL of suspension. An equal volume of the MNase digestion buffer (0.02 U/µL MNase (Thermo Scientific, Cat. No: 88216) in 2x lysis buffer was added to the cell suspension and gently vortexed. The cell suspension was then incubated on ice for 20 minutes, followed by incubation at 55°C water bath for 10 minutes. The digestion reaction was terminated using 30 mM EGTA. Then the digested chromatin was protease k (Ambion™) treated at 55°C and then pre-cleared with 1:2 KAPA Pure beads (Roche Cat. No: 07983298001). The quality and quantity of fragmented DNA were verified using agarose gel (1.5%) electrophoresis and Qubit® 4.0 Fluorometer (Thermo Fisher Scientific, Waltham, Massachusetts, U.S), respectively. Illumina KAPA LTP Library Preparation Kit was employed for library construction (Cat. No: KK8233). Purified, fragmented DNA was end-repaired to produce 5′-phosphorylated and blunt-ended dsDNA fragments. Unique Dual Indexes (UDIs) were ligated to 3'-A-tailed library fragments holding appropriate adapter sequences on both ends. DNA fragments were subjected to methylated DNA immunoprecipitation, followed by library amplification.