RRHP 5-hmC Library Prep Kit

D5450 / D5451

RRHP 5-hmC Library Prep Kit

D5450 / D5451

Cat # Name Size Price Quantity
D5450 RRHP 5-hmC Library Prep Kit 12 Preps $990.00
+ -
D5451 RRHP 5-hmC Library Prep Kit 25 Preps $1,680.00
+ -


RRHP 5-hmC Library Prep Kit


  • 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).


The RRHP 5-hmC Library Prep Kit is an all-inclusive solution for analysis of genome-wide 5-hydroxymethylcytosine (5-hmC) positions at single-base resolution. 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.

Technical Specifications

Equipment Required Microcentrifuge, thermocycler with heated lid, and gel electrophoresis or other equipment for DNA visualization
Input 100 ng - 1 µg
Sample Source High quality, intact genomic DNA free of enzymatic inhibitors.
Sequencing Compatibility Any Illumina sequencing platforms

Product FAQ

Q1: Can RRHP be used for region specific detection of 5-hmC?

Q2: Why do I see my negative control appear after library amplification?

Q3: Are the index primers used in the RRHP 5-hmC Library Prep Kit (D5451) the same as in the Pico Methyl-Seq Library Kit (D5455)?

Q4: How should I trim and align the sequencing data?

Q5: What instruments are the libraries compatible with and what sequencing parameters should I use?

Q6: What are some tips to ensure optimal library yield?

Q7: What is the concentration of the supplied index primers?

Q8: Are index primers provided in other company’s library preparation kits compatible with the Zymo adapters?

Q9: I need additional index primers. Can I purchase or synthesize them?

Q10: Can beads be used to perform the size-selection instead of an agarose gel (section IV)?

Q11: Can the final MspI digestion step be increased?

Q12: Is the Glucosylation reaction (step 6) complete after 2 hours?

Q13: Should MspI be heat-inactivated after initial digestion?

Q14: What is the optimal DNA input amount?

Q15: Can both methylation and hydroxymethylation be detected using RRHP?

Q16: Is RRHP a quantitative method? How does it differ from TAB-seq and oxBS-seq, which also looks at 5hmC?

Q17: Can a different polymerase than the 2x QuestTaq Master Mix be used?


Characterization of midbrain dopaminergic neurons is important for understanding Parkinson’s disease, but identifying and purifying induced dopaminergic (iDA) neurons from a heterogeneous cell population can be difficult. Therefore, the authors generated an embryonic stem cell line with a tyrosine hydroxylase (TH)-RFP reporter to selectively purify iDA neurons, enabling precise transcriptional and epigenetics analysis. The 5-hydroxymethylation patterns profiled using the RRHP kit were compared to identified enhancer regions and RNA-seq data. The analysis revealed dozens of transcription factors, including homeobox TF UNCX and other well-known dopaminergic neuron regulators, that may be important for induction and maintenance of dopaminergic neurons.

Xia N, et. al. (2017) A Knockin Reporter Allows Purification and Characterization of mDA Neurons from Heterogeneous Populations. Cell Reports 18(10)2533-2546.

Kit Components