Anti 5 methylcytosine monoclonal antibody

For each developmental stage, the tooth germs were isolated separately from three randomly selected embryos with a specific sex (male) as biological replicates. MeDIP DNA libraries were constructed following the protocol as described previously [47 (link)]. In brief, genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Five microgram DNA was sonicated to fragments ranging from 100 to 500 bp. Subsequently, DNA underwent end-repair, the generation of 3’-dA overhangs, and adaptor ligation steps using Paired-End DNA Sample Prep kit (Illumina, San Diego, CA, USA). Adaptor-ligated DNA was then immunoprecipitated by anti-5-methylcytosine monoclonal antibody (Diagenode, NJ, USA). The enriched methylated fragments and 10 % input DNA were purified on DNA Clean & Concentrator-5 columns (Zymo, CA, USA) according to the manufacturer’s manuals. Enriched fragments were amplified by adaptor-mediated PCR, the products were quantified on Agilent 2100 Analyzer (Agilent Technologies, Santa Clara, CA, USA), and MeDIP library was sequenced on an Illumina HiSeq 2000 Sequencing System by Beijing Genomics Institute (BGI, Shenzhen, China). After the completion of sequencing run, raw data was processed by the Illumina base-calling pipeline.