The EpiNext™ In Situ Accessible Chromatin (ISAC) Preparation Kit is a complete set of optimized reagents designed for the preparation of in situ accessible chromatin to be used for identifying in situ chromatin accessibility by gene-specific analysis via real-time PCR or genome-wide analysis via next generation sequencing, which allows for open/closed chromatin status profiling and determination of nucleosome/transcription factor positioning from biological samples. The kit has the following advantages and features:
Background Information Determination of chromatin accessibility through identifying open/closed chromatin and mapping the nucleosome positioning along the genome is directly linked to epigenetic gene regulation and chromatin states and structure. Several methods are currently used to detect chromatin accessibility. The traditional method involves employing DNase I or micrococcal nuclease (MNase) combined with NGS (DNase I-seq or MNase-seq, respectively) as tools to assess chromatin accessibility. However, using DNase I or MNase has 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, these methods need to use isolated chromatin, which may not preserve native chromatin structure and spatial organization of regulatory elements within a cell, thereby potentially disrupting important chromatin interactions and altering accessibility patterns. Using transposase such as Tn5 transposase to cleave accessible chromatin combined with next generation sequencing (ATAC-seq) is a technique that was first described as an advanced alternative method to MNase-seq and DNase-seq. Using hyperactive mutant Tn5 transposase that inserts sequencing adapters into open chromatin regions in an in situ status is shown to be faster and more sensitive in epigenome analysis than DNase-seq or MNase-seq and needs much less starting material amount. However, it has significant fragmentation and GC content bias because of sequence-specific cleavage of Tn5 enzyme. Also, it is disadvantaged by covering less accessible chromatin sites compared to MNase and DNase.
Principle & Procedure This kit contains all the necessary reagents required for preparing in situ accessible chromatin from cell/tissue samples. In this assay, cells are directly exposed to a unique nucleic acid cleavage enzyme mix after permeabilization. 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 protein/histone is unaffected. DNA is then purified from target chromatin for use in gene-specific analysis via real-time PCR or genome-wide analysis via NGS.
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