RRHP 5-hmC Library Prep Kit
D5450 / D5451
- 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).
|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|
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.