D546 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 of 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 percentage (≥70% if using human genomic DNA) of all CpG islands, promotors, and gene bodies while largely reducing the sequencing cost, making it ideal for high-throughput screening and comparison experiments.
Order D546 Zymo-Seq RRBS Library Kit in USA or Canada from MBP Inc. for fast delivery and competitive prices.
| Equipment | Microcentrifuge, thermocycler |
|---|---|
| Input | 10 pg – 100 ng |
| Sample Source | The protocol is designed for 10 pg – 100 ng genomic DNA input. 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 (D4010). |
| Sequencing Compatibility | This kit produces libraries with Illumina TruSeq Adapters that can be sequenced on any of the Illumina sequencing platforms. |
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: How many libraries can I multiplex and sequence in a single lane using this kit?
We provide 12 pre-mixed Unique Dual Index (UDI) primer sets with the kits and therefore you can multiplex up to 12 libraries. If you want to multiplex more, please order the Zymo-Seq UDI Primer Plate (Indexes 1-96, Cat. No. D3096) separately.
Q7: 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.
Q8: 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.
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