New England Biolabs NEBNext Enzymatic 5hmC-seq kit

Monday, 01 April, 2024 | Supplied by: New England Biolabs

New England Biolabs NEBNext Enzymatic 5hmC-seq kit

The NEBNext Enzymatic 5hmC-seq (E5hmC-seq) kit represents an advancement in the field of epigenetic research. Historically, the precise identification of 5-hydroxymethylcytosine (5hmC) at single-base resolution has been a challenging task, largely due to limitations in existing sequencing technologies. The kit effectively bridges this gap, enabling researchers to comfortably differentiate 5hmC from 5-methylcytosine (5mC).

Central to its innovation is the novel enzymatic conversion process. Initially, 5hmC is glucosylated using T4-BGT. This is followed by deamination of 5mC and unmodified cytosine to thymine and uracil, respectively, via APOBEC, leaving 5hmC untouched. This process enables Illumina sequencing to represent 5hmCs as cytosine and distinguish them from other forms.

The E5hmC-seq kit accommodates a broad range of DNA input (100 pg to 200 ng), enabling high-quality data even at lower concentrations, in an improvement over traditional methods. Its compatibility with mechanical fragmentation techniques, like Covaris, optimises DNA preparation, while specific recommendations for NEBNext UltraShear are provided to maintain methylation marks. Additionally, the workflow includes NEBNext Ultra II DNA reagents for end repair and dA-tailing, and a specialised E5hmC-seq Adaptor paired with a modified Q5 High Fidelity DNA Polymerase (Q5U), optimising library amplification.

Designed for seamless integration with all Illumina sequencing platforms, NEBNext Enzymatic 5hmC-seq empowers researchers with the ability to unravel the complexities of epigenetic regulation, particularly the role of 5hmC in various biological processes and diseases. The kit is set to be a useful tool in the advancement of epigenetic research and DNA methylation analysis.

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