SAVE 20% ON RRBS KITS FROM DIAGENODE!
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Jun 24 2020
Aug 1 2020
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Save on Reduced Representation Bisulfite Sequencing (RRBS) Kits!
DNA methylation is a key epigenetic mechanism with important regulatory functions in development and disease. Bisulfite sequencing enables the detection of cytosine methylation at single-base resolution. Nowadays, this method is widely used for targeted gene analysis due to a relatively low price per reaction. However very few researchers perform genome-wide studies, discouraged by the enormous sequencing efforts and corresponding costs. Reduced Representation Bisulfite Sequencing (RRBS) is the best alternative to increase the scale of the analysis cost efficiently. In the initial step of the RRBS protocol, DNA is digested with the restriction enzyme MspI wich recognizes CCGG sites. Thus, all resulting genomic fragments will start and end with a CpG dinucleotide. Due to the biased distribution of CpGs throughout the genome (depicted in Figure 1), MspI digestion followed by size selection enriches for the most CpG-rich regions, including CpG islands. DNA methylation at these regulating regions, which are often located at gene promoters, is most likely to influence gene expression. Thus, RRBS provides a cost-effective method to analyze DNA methylation in the most relevant genomic regions.
Diagenode’s Premium RRBS kit offers a complete solution, including reagents for enzymatic digestion, library preparation, bisulfite conversion and amplification. Moreover this protocol requires only very little starting material (100 ng of gDNA is recommended) and is optimized for high throughput and sequencing on Illumina platforms. Since six samples can be pooled together and processed simultaneously, an experiment starting with 96 samples will require handling only 16 tubes for the bisulfite conversion and only 16 sequencing lanes. Handling time, amount of reagents, and the price per sample are thus greatly reduced.
- Positive and negative spike-in controls are included for the monitoring of bisulfite conversion efficiency
- Size selection has been optimized to keep small fragments of interest and to remove adaptor dimers, resulting in a better coverage
- The pooling protocol includes a quantification of the samples and a pooling application, available on our website, to help you to group your samples, depending on the DNA amount and adaptor barcode of each sample
- The bisulfite conversion protocol has been improved to decrease DNA degradation while keeping a highly efficient conversion of unmethylated cytosines
- The minimum number of amplification cycles needed for each pool is determined to avoid amplification biases. Our MethylTaq Plus enzyme was developed to amplify bisulfite converted DNA with high efficiency, and reduces the number of PCR cycles required
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