hMeDIP of mus musculus: corneal epithelium, 2 months
To prepare hMeDIP-seq libraries, 1 g of gDNA was fragmented to 350bp (Covaris M220, Covaris, LLC, Woburn, MA, USA). The fragmented DNA was cleaned, denatured, and resuspended in immunoprecipitation buffer. 2 g of a 5hmC-specific antibody (#39791, Active Motif, Carlsbad, CA, USA) was added for immunoprecipitation (IP) overnight at 4C. 10% of the DNA being used in the IP reaction was saved for use as Input (IN) DNA. Protein G magnetic beads were used to perform the pull-down of 5hmC-enriched fragments. The 5hmC-enriched DNA fragments (IP) were then released from the antibody by digestion with Proteinase K. After cleanup with AMPure XP beads, the 5hmC-enriched DNA fragments (IP) and Input (IN) DNA were used to prepare libraries using the NEBNext Ultra II DNA Library Prep with Sample Purification Beads (E7103S, New England Biolabs, Ipswich, MA, USA). The hMeDIP-seq libraries were quantified using Qubit 4 Fluorometer, and sizes were evaluated using the 4200 TapeStation system.
SUB14819163
CE14m_1_hMeDIP_IP_O1