EpiNext CUT&LUNCH-Seq Kit

The EpiNext™ CUT&LUNCH (Cleavage Under Target and Liberate Unique Nucleic Complex Homogeneously)-Seq Kit is a complete set of optimized reagents designed for, in a fast manner, enriching a protein (histone or strong binding transcription factor)-specific DNA complex and preparing DNA library from low input cells to profile interactions between proteins and DNA via next generation sequencing using Illumina platforms. The kit has the following advantages and features:

• Extremely Fast Protocol:Start directly from intact cells without requiring ConA immobilization. Complete the entire procedure—from cell sample to ready-to-use library DNA—in under 3 hours, minimizing nuclear damage and loss of disassociated chromatin components, preserving chromatin structure, and reducing loss of valuable samples.
  
• Enhanced Specificity and Minimized Background: Unique nucleic acid cleavage enzymes ensure low sequence bias by selectively cleaving DNA at both ends of the target protein/DNA complex without affecting the DNA bound to the protein. Non-specific protein-DNA complexes are selectively eliminated, enabling high-resolution mapping of target protein-enriched regions.
• Low Input Materials: Robust unbound DNA cleavage and immunocapture are completed in minimal time. This method uses intact cells while providing maximal degradation protection of the target protein and minimal sample loss. As a result, the input cell amount can be as few as 2000 cells.
  
• Wide Suitability: Suitable for cultured cells from a flask or plate, primary cells, or rare cell populations isolated from blood, body fluid, specific cells sorted from entire cell populations, embryonic cells, etc. It is compatible with histones and transcription factors while maintaining assay specificity and sensitivity.
  
• Highly Convenient: The kit includes all necessary components, making the EpiNext™ CUT&LUNCH-Seq Kit both convenient and cost-effective.
• Seamless Integration with Existing Pipelines: Enriched DNA for NGS can be analyzed with existing, time-tested ChIP-seq bioinformatics software and tools.

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CUT&LUNCH histone Marker peak profiles are consistent with ENCODE ChIP-seq profiles. CUT&LUNCH: Peak scores are plotted in the bar graph for 0.1 million MCF-7 cells. ENCODE ChIP-Seq: Peak scores are plotted in the bar graph for various cells (5-10 million) including MCF-7 cells. A: H3K4me3 (active regions), B: H3K9me3 in MCF-cells (silenced regions).

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CUT&LUNCH TF (RNA polymerase II) peak profiles are consistent with ENCODE ChIP-seq profiles. CUT&LUNCH: Peak scores are plotted in the bar graph for 0.1 million MCF-7 cells. ENCODE ChIP-Seq: Peak scores are plotted in the bar graph for 5 million cells.

Background Information
Enrichment of histone or transcription factor (TF)-complexed DNA in vivo, followed by qPCR and/or next-generation sequencing (NGS), offers an advantageous tool for studying genome-wide protein-DNA interactions. The major method commonly used to achieve this goal is chromatin immunoprecipitation followed by sequencing (ChIP-seq). This method includes highly specific ChIP-exo and ChIP-nexus. These two assays provide high-resolution mapping. However, the major limitation of these assays is that they need a large amount of input material to produce a strong enough signal over background noise. CUT&RUN (Cleavage Under Targets & Release Using Nuclease) and CUT&TAG (Cleavage Under Targets & Tagmentation) were developed for mapping protein-DNA interaction with limited biological materials, which require much less sample amount and also significantly improve mapping resolution in NGS. However, a considerable drawback of these assays is non-specific cleavage by antibody un-coupled pAG-MNase, significantly limiting the specificity of the CUT&RUN use for most transcription factors in different species and cell types. In addition, these assays have complicated steps and need to optimize assay conditions, which is still time-consuming.

Principle & Procedure
This kit contains all the necessary reagents required for enriching a protein (histone or strong binding transcription factor)-specific DNA complex for NGS library preparation, starting from cells. In this assay, cells are permeabilized and exposed to the ChIP-grade antibody of interest. With the use of a unique nucleic acid cleavage enzyme mix, DNA sequences at both ends of the target chromatin regions are cleaved/removed. The liberated non-specific protein-DNA complexes are eliminated, and only antibody-bound complexes will be selectively recovered. The adaptors are ligated to the target DNA fragments purified from the captured protein/DNA complex. The ligated DNA is then amplified with a high-fidelity PCR mix for library DNA construction to profile interactions between proteins and DNA using NGS.

Starting Materials
Starting materials can include various mammalian cell samples such as cells from a flask or microplate cultured cells.