Zymo-Seq RRBS Library Kit

EZ DNA Methylation RRBS Library Prep Kit


  • Simple workflow: Prepare Reduced Representation Bisulfite Sequencing (RRBS) libraries in as little as 2 hours of hands-on time.
  • Low input: The only RRBS kit that produces NGS libraries from = 10 ng of genomic DNA.
  • Accurate and reproducible: Unbiased methylation calling and reproducible CpG coverage.


Zymo-Seq RRBS Library Kit is an all-in-one RRBS library prep kit that generates NGS libraries with a simplified workflow for the classic method Reduced Representation Bisulfite Sequencing (RRBS). This all-in-one RRBS library prep kit allows for DNA methylation profiling at single-nucleotide resolution in CpG-rich regions of the genome. This Zymo-Seq RRBS Library Kit is the ideal RRBS library prep kit to generate libraries that cover a significant percent (=70% if using human genomic DNA) of all CpG islands, promotors, and gene bodies while largely reduces the sequencing cost, making it ideal for high-throughput screening and comparison experiments.

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Technical Specifications

Equipment Required Microcentrifuge, thermocycler with a heated lid
Input 10 – 500 ng of genomic DNA
Sample Sources Purified genomic DNA. The DNA should be free of enzymatic inhibitors and can be suspended in water, TE, or a low salt buffer. DNA with low 260/280 or 260/230 ratios should be purified prior to processing using the Genomic DNA Clean & Concentrator (Cat. No. D4010). This protocol is not recommended for samples from species with low CpG density.
Sequencing Platform Compatible Libraries are compatible with all Illumina sequencing platforms except HiSeq X Series.

Product FAQ

Q1: Does the Zymo-Seq RRBS Library Kit offer all the necessary reagents to generate the libraries for Reduced Representation Bisulfite Sequencing?

Yes. With this all-in-one kit, you can generate RRBS libraries with the genomic DNA of your interest. However, the reagents for library quantification and size distribution characterization are not provided and will need to be supplied by the customers and/or their sequencing service providers to ensure optimal practice for sequencing.

Q2: Is there any restriction on the sample species when using this kit?

In principle, the Zymo-Seq RRBS Library Kit can be applied to genomic DNA from any species, as long as a reference genome exists for subsequent bioinformatic analysis and the genomic DNA is between 10 and 500 ng with high quality (a good rule of thumb for “high quality” is that the DNA is of little degradation and the majority of the content being higher than 10 kb).

Q3: Can I use less than 10 ng of genomic DNA with this kit?

It is possible to generate libraries with less than 10 ng of genomic DNA; however, the quality of such libraries is not guaranteed.

Q4: Can I use DNA extracted from FFPE samples to generate RRBS libraries with this kit?

It is possible to generate libraries with DNA extracted from FFPE samples. However, due to the degradation of such DNA, the quality of the libraries will be affected. Increasing the input amount may help but the quality of the libraries is not guaranteed.

Q5: What method of quantification is recommended for the input DNA?

We quantify input DNA using Nanodrop or Qubit, but any method should do. The input genomic DNA should be RNA-free.

Q6: Are the index primer sets used in RRHP 5-hmC Library Prep Kit (D5450, D5451) and Pico Methyl-Seq Library Prep Kit (D5455, D5456) the same as those used in Zymo-Seq RRBS Library Kit (D5460, D5461)?

Yes, all these library prep kits use the same index primer sets.

Q7: How many libraries can I multiplex and sequence in a single lane using this kit?

We provide 12 single-indexed primer sets with the kits and therefore you can multiplex up to 12 libraries. If you want to multiplex more, or need dual indexing, please contact our Technical Support for assistance ((949) 679-1190 ext. 3 or tech@zymoresearch.com)

Q8: What is the expected yield of each library generated using this kit?

The yield per library depends on the quality of the input genomic DNA and other factors. In general, the yield of each library is around 300 ng or more (if quantified with Nanodrop) when following the recommended PCR cycles in the instruction manual.

Q9: What read length and sequence depth should I seek for when sequencing RRBS libraries?

The optimal read length and sequence depth depend on the goal of the project and the subsequent bioinformatic analysis. In general, when handling mammalian samples, 50-bp single-end or paired-end sequencing should do as the RRBS libraries usually have comparatively short inserts. Longer read length is also possible if one wants to cover as many CpG sites as possible. Additionally, a 5-10x mean coverage per CpG site is recommended.



“The Zymo-Seq RRBS Kit is impressive in its simplicity. This kit empowers researchers to interrogate genomic DNA methylation with an easy-to-use protocol and comparatively little hands on time, and yields robust and reproducible results. Compared to other RRBS library preparation methods, this is by far the easiest protocol I have come across: a researcher who has never done a library preparation or who is new to researching DNA methylation could use this kit with confidence. We were further impressed with the kit’s ability to tolerate low inputs down to 10ng, and because of this we were able to confidently assess methylation patterns in the embryonic chicken using minimal DNA input. In the past we have used whole genome bisulfite sequencing to investigate DNA methylation, but with the availability of this kit, we have turned our attention to RRBS as a cost-effective alternative that does not have the restriction of needing a large amount of DNA input to yield sufficiently complex libraries. In the future we are very likely to purchase this kit for use in other projects, including applications to human iPSC cultures, avian, and amphibian models.”

– – Jared T. (Miami University)

Kit Components

Zymo-Seq RRBS Library Kit

D5460 / D5461

Cat # Name Size
D5460 Zymo-Seq RRBS Library Kit (24 preps) 24 Preps
D5461 Zymo-Seq RRBS Library Kit (48 preps) 48 Preps