GSM5419627: 1KO-5mC_G2; Mus musculus; MeDIP-Seq
One μg of genomic DNA sheared to 200-500 bp using the LE220 Focused-ultrasonicator (Covaris) was end-repaired and A-tailed. The Illumina PE adapter-ligated DNA was heat-denatured and applied to MeDIP overnight with a monoclonal antibody for 5mC (AnaSpec, Inc.). hMeDIP was performed using a rat monoclonal antibody (Diagenode) for 5hmC. The IP material was recovered with a secondary antibody of rabbit and rat anti-mouse IgG, respectively (Jackson ImmunoResearch) along with protein A/G (Thermo Fisher Scientific), followed by digestion with 20 mg/ml Proteinase K (Invitrogen) for 2 hours. IP DNA was purified using the MinElute PCR Purification Kit (Qiagen). Confirmation of concentration of 5mC and 5hmC DNA was evaluated by qPCR using positive and negative control primers. The final library amplification was performed using Phusion® hot start II DNA polymerase and 5× Phusion® GC Buffer (Thermo Fisher Scientific) with Illumina paired-end index primers. The PCR-amplified library was size-selected for 220–450-bp fragments on an 8% PAGE gel. The library was normalized based on molarity and pooled for sequencing using v4 ready-to-load reagents for HiSeq 2500 System (Illumina) at 75-bp paired-end reads.