The Reduced Representation Hydroxymethylation Profiling (RRHP) method is based on blocking MspI digestion by glucosylating 5-hmC within MspI recognition sites. Fragments lacking glucosylated 5-hmC at the adapter-ligation junction will be cleaved and not amplified by PCR. Therefore, only fragments containing 5-hmC will be successfully amplified and analyzed by Next-Generation Sequencing. Fragments with higher 5-hmC levels will be correlated with higher frequency of sequencing reads. RRHP bypasses the need for bisulfite conversion, which allows for DNA inputs as low as 100 ng, lower sequencing depth, and straight-forward bioinformatics processing.
HIGHLIGHTS:
·Innovative library preparation for strand-specific mapping of 5-hmC in DNA.
·Streamlined workflow accommodates low (≥100ng) DNA inputs.
·Libraries are ready for Next-Generation sequencing (Illumina-compatible).