RRHP™ 5-hmC Library Prep Kit

Innovative library preparation for strand-specific mapping of 5-hmC in DNA.
Streamlined workflow accommodates low (≥100 ng) DNA inputs.
Libraries are ideal for Next-Gen sequencing or array.

Product Size Catalog # Price Qty
RRHP™ 5-hmC Library Prep Kit 12 Preps D5450
$990.00
RRHP™ 5-hmC Library Prep Kit 25 Preps D5451
$1,680.00

About RRHP™ 5-hmC Library Prep Kit

The RRHP™ 5-hmC Library Prep Kit is the first comprehensive solution for quantitative analysis of genome-wide 5-hmC positions at single base resolution. The Reduced Representation Hydroxymethylcytosine Profiling(RRHP) system is based on blocking MspI digestion by glucosylating 5-hmC within MspI recognition sites. Shown in the schematic below, fragments lacking glucosylated-5-hmC at the adapter-ligation junction will be cleaved and not amplified by PCR. Positive selection relies on fragments containing 5-hmC being successfully amplified and analyzed. 


Sample Sources Input ≥100 ng intact genomic DNA. DNA can be in water, TE, or low salt buffer and should be free of enzymatic inhibitors.
Equipment Needed Microcentrifuge and a heat block, water bath, or thermal cycler for heat steps. A thermal cycler is required for PCR and electrophoresis equipment for DNA selection.
Sequencing This system is designed for preparation of libraries compatible with Illumina’s TruSeq chemistries for HiSeq™ and MiSeq™ platforms.

Single-Site Display of 5-hmC. Human brain DNA was prepared using the RRHP™ 5-hmC Library Prep Kit and then sequenced with an Illumina HiSeq™ 2000 with 50 bp single-end reads. Sequence was aligned to the human reference genome. Tracks (bottom figure) display relative quantitation of 5-hmC strand distribution (blue = sense, red = antisense) as well as SNP positions within the fragments at a single locus.

Section I: Digestion of Genomic DNA  

  1. Add the following to a PCR tube: 
  2.                      Genomic DNA (≤ 1 μg)                                          X μl

                         10X RRHP Reaction Buffer                                 5 μl

                         MspI (20 U/μl)                                                     1.5 μl

                         H2O                                                                          Y μl 

                                                                                                        50 μl

                  Note: To ensure complete digestion, we recommend the input DNA does not exceed 1 μg per reaction.

  3. Mix, spin briefly, and then incubate the reaction at 37 °C for 8 hours.
  4. Transfer the digestion to 300 μl DNA Binding Buffer in a Zymo-Spin IC Column in a Collection Tube. Spin at ≥ 10,000 x g for 30 seconds.
  5. Discard the flow-through. Add 200 μl DNA Wash Buffer to the column. Spin at ≥ 10,000 x g for 30 seconds. Repeat this wash step.
  6. Transfer the Zymo-Spin IC Column to a 1.5 ml microcentrifuge tube. Add 15 μl DNA Elution Buffer directly to the column matrix and wait for 1 minute. Spin at ≥ 10,000 x g for 1 minute to elute the digested DNA.
  7.  

    Section II: Adapter Ligation, Extension, and Library Glucosylation

  1. Add the following to a PCR tube:
  2.                       Digested DNA                                                15 μl

                         10X RRHP Reaction Buffer                            2 μl

                         T4 DNA Ligase (400 U/μl)                               1 μl

                         RRHP Adapter 1 (20 μM)                             0.5 μl

                         RRHP Adapter 2 (20 μM)                             0.5 μl

                         rATP (10 mM)                                                0.5 µl

                         H2O                                                                  0.5 μl

                                                                                                   20 μl

  3. ix, spin briefly, and then incubate at 16 °C for 2 hours.
  4.              Note: If desired, adapter ligation can be carried out overnight at 16 °C without diminishing the quality of the library.

  5. Following the incubation, add the following to the tube:
  6.                     Taq DNA Polymerase (2 U/μl)                   0.5 μl

                        dNTPs (10 mM; 2.5 mM/ea)                       1.5 μl

                                                                                                  22 μl total volume

  7. Mix briefly, spin, and then incubate at 72 °C for 30 minutes in a thermal cycler with a heated lid. Hold at 4 °C.
  8.  Following the incubation, add the following to the tube:
  9.                    10X RRHP Reaction Buffer                            3 μl

                       10X UDPG (1 mM)                                            5 μl

                       β-Glucosyltransferase (2 U/μl)                     3 μl

                       H2O                                                                   17 μl 

                                                                                                  50 μl total volume

  10. Incubate the reaction at 37 °C for 2 hours.
  11. Transfer the reaction to 500 μl DNA Binding Buffer in a Zymo-Spin IC Column in a Collection Tube. Mix thoroughly, and then spin at ≥ 10,000 x g for 30 seconds.
  12. Discard the flow-through. Apply 200 μl DNA Wash Buffer to the column and then spin at ≥ 10,000 x g for 30 seconds. Repeat this wash step.
  13. Transfer the Zymo-Spin IC Column to a 1.5-ml microcentrifuge tube.  Apply 15 μl DNA Elution Buffer directly to the column matrix and wait for 1 minute. Spin at ≥ 10,000 x g for 1 minute to elute the glucosylated DNA.
  14.  

    Section III: Final Digestion

  1. Add the following to a PCR tube:
  2.                     Glucosylated DNA                                   15 μl

                        10X RRHP Reaction Buffer                     3 μl

                        MspI (20 U/μl)                                          1.5 μl

                        H2O                                                          10.5 μl 

                                                                                             30 μl

  3. Mix, spin briefly, and then incubate at 37 °C for 3 hours.
  4. Transfer the reaction to 65 °C for 15 minutes for complete enzyme inactivation. Add loading dye (1X final concentration) and then store on ice until the next step.
  5.  

    Section IV: Size Selection

  1. Proceed with size selection*. For the best resolution, use a 2% (w/v) agarose/TAE gel w/EtBr. Gels should be electrophoresed at 110 V such that the range from 100-500 bp can be confidently isolated in an excised gel slice.
  2. Add each excised gel slice to 3 mass volumes ADB Buffer (1:3) in a 1.5 ml microcentrifuge tube. Incubate at 50 °C for at least 30 minutes. Vortex the mixture periodically to ensure complete solubilization of the gel matrix.
  3. Transfer to a Zymo-Spin IC Column in a Collection Tube. If the total volume exceeds 800 μl, load the column twice. Spin at ≥ 10,000 x g for 30 seconds.
  4. Discard the flow-through. Add 300 μl DNA Wash Buffer to the column. Spin at ≥ 10,000 x g for 30 seconds. Repeat this wash step.
  5. Transfer the Zymo-Spin IC Column to a 1.5-ml microcentrifuge tube. Add 15 μl DNA Elution Buffer directly to the column matrix and wait for 1 minute. Spin at ≥ 10,000 x g for 1 minute to elute the size-selected DNA library.
  6.  

    Section V: Library Enrichment with Limited Amplification

    The kit supplies six (6) Index Primer Sets for barcoding different samples for multiplexed sequencing. The index information is provided in the table below. Make sure to use a primer set only once per single lane.

    Index Primer Set

    Standard Illumina Designation

    Index Sequence

    A

    2

    CGATGT

    B

    4

    TGACCA

    C

    5

    ACAGTG

    D

    6

    GCCAAT

    E

    7

    CAGATC

    F

    12

    CTTGTA

     

  1. Assemble the following components in a PCR tube:
  2.                     Size-selected DNA library                               15 μl

                        Index Primer Set (10 μM/ea)                         1 μl

                        2X QuestTaq Master Mix                           25 μl

                        H2O                                                                       9 μl 

                                                                                                    50 μl

                Cycle with the following thermal profile:

    95 °C     3 min  
    95 °C     30 sec  
    59 °C     30 sec     --> 10 cycles
    72 °C     30 sec  
    72 °C     1 min  
    4 °C     hold
  3. After the first 10 cycles, sample 5 μl of the reaction mix for analysis by agarose gel electrophoresis. Cycling is complete upon visualization of a faint product between 150-500 bp*.  If no visible product appears in the gel, add an additional 4 cycles until a product does appear. 
  4. Library Enrichment by Limited Amplification.  Human brain DNA was fragmented with MspI then ligated together with RRHP adapters to create sequencing libraries. When glucosylated (libraries 3-6), the library is resistant to subsequent MspI or HpaII digestion and can be amplified. Non-glucosylated samples (libraries 2 and 7) are susceptible to digestion and cannot be amplified for library enrichment. Digestion with HpaII, an isoschizomer of MspI with methylation sensitivity, allows for inclusion of methylated fragments in the final library display.

  5. After cycling is complete, add 5 volumes of DNA Binding Buffer to the reaction (5:1) and transfer to a Zymo-Spin IC Column in a Collection Tube. Spin at ≥ 10,000 x g for 30 seconds.
  6. Discard the flow-through from the Collection Tube and apply 200 μl DNA Wash Buffer to the column. Spin at ≥ 10,000 x g for 30 seconds. Repeat this wash step.
  7. Transfer the Zymo-Spin IC Column to a 1.5-ml microcentrifuge tube. Add 15 μl DNA Elution Buffer directly to the column matrix and wait for 1 minute. Spin at ≥ 10,000 x g for 1 minute to elute the completed DNA library.
For specific notes and additional information, please see the product protocol PDF.
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