EpiNext Chromatin Accessibility Sequencing Fast Kit

Product Description

The EpiNext™ Chromatin Accessibility Sequencing Fast Kit is a complete set of optimized reagents designed for conducting a genome-wide analysis of chromatin accessibility, including nucleosome/transcription factor positioning from various biological samples via next-generation sequencing.  The kit has the following advantages and features:

• Extremely fast three-step protocol: Completes the procedure (from cell/tissue sample to sequencing-ready library DNA) in just 1 hour and 55 minutes, faster than ATAC-seq. Minimizes nuclear damage, preserves chromatin structure, and avoids mitochondrial contamination
• Versatile usage: Works with small cell amounts of fresh/frozen tissues, even in harsh environments, unlike DNAse-seq/MNase-seq
• Reduced biases: Absence of sequence-specific cleavage minimizes fragmentation bias and biases related to GC or AT content
• Comprehensive kit: Includes all necessary components for each step, ensuring convenience and cost-effectiveness of the EpiNext™ Chromatin Accessibility Sequencing Fast Kit

Background Information

Determination of chromatin accessibility through mapping of the nucleosome positioning along the genome is directly linked to epigenetic gene regulation and chromatin states and structure. There are several methods currently used for detecting chromatin accessibility. The traditional method involves employing DNase I (DNase-seq) or micrococcal nuclease (MNase-seq) as tools to assess chromatin accessibility. However, these methods may have drawbacks that limit them from broad-spectrum use. They require a significant amount of starting material and are susceptible to assay bias due to challenges regulating enzyme concentrations and digestion duration. Moreover, they are unable to function in a harsh environment and are unsuitable for tissue samples. The assay for transposase-accessible chromatin with next generation sequencing (ATAC-Seq) is a technique that was first described as an alternative advanced method for MNase-seq, FAIRE-seq, and DNase-seq. In ATAC-seq, intact nuclei are directly used, and accessible DNA regions are identified by probing open chromatin with hyperactive mutant Tn5 transposase that inserts sequencing adapters into open chromatin regions. This method is shown to be faster and more sensitive in epigenome analysis than DNase-seq or MNase-seq. However, ATAC-seq has significant fragmentation and GC content bias because of sequence-specific cleavage of Tn5 enzyme. Additionally, it is disadvantaged by the prevalence of mitochondrial contamination due to non-specific insertion of DNA into both mitochondrial DNA and nuclear DNA.

Principle & Procedure

The EpiQuik™ Chromatin Accessibility Assay Kit contains all the necessary reagents required for obtaining a genome-wide analysis of chromatin accessibility from cell/tissue samples via NGS. In this assay, nuclei are isolated from the cells/tissues and are exposed to a unique nucleic acid cleavage enzyme mix. Chromatin is fragmented, and DNA sequences at both ends of the target chromatin are cleaved/removed. At the same time, the DNA sequence occupied by the target protein/histone is unaffected. The adaptors are ligated to the target protein/histone-bound DNA fragments. The ligated DNA is then released, purified, and amplified with a high-fidelity PCR mix for library DNA construction. 

Starting Materials & Input Amount

Starting materials can include various mammalian tissue or cell samples such as cells from flask or microplate cultured cells, fresh and frozen tissues, etc. The amount of cell/tissues for each reaction can be from 1 x 10⁵ cells or 2 mg tissues to 2 x 10⁶ cells or 40 mg tissues. For an optimal reaction, the input chromatin amount should be about 0.5 x 10⁶ cells or 10 mg tissues.

Performance Data

Fig.2. Characterization of EpiNext™ Chromatin Accessibility Sequencing DNA library. Chromatin isolated from 100,000 MCF-7 cells was cleaved and used for DNA library preparation with the EpiNext™ Chromatin Accessibility Sequencing Fast Kit. The library was analyzed with a Bioanalyzer®. The representative library contains approximately 170 – 800 bp fragments with peak at around 320 bps as shown on the electropherogram.