EpiNext CUT&RUN Fast Kit | P-2028

The EpiNext™ CUT&RUN Fast Kit is a complete set of optimized reagents required for carrying out a successful CUT&RUN starting from mammalian cells or isolated nuclei/chromatin. The kit is designed to quickly enrich specific DNA complexes with a protein (histone or transcription factor) from low input cells/chromatin and to identify or map in vivo protein-DNA interaction by next generation sequencing using Illumina platforms or other methods such as qPCR. The innovative working principle, optimized protocol, and components of the kit allow for the capture of targeted protein/DNA complexes with minimized non-specific background levels. The captured DNA is specifically suitable for the construction of both non-barcoded (singleplexed) and barcoded (multiplexed) libraries to map target protein-DNA interaction regions with less bias and at a high resolution.

• High Enrichment: Uses a unique nucleic acid cleavage enzyme mix, which has low sequence bias, to simultaneously fragment chromatin and cleave/remove any DNA sequences in both ends of the target protein/DNA complex without affecting DNA occupied by the target protein. The enriched DNA >20 bps can be efficiently and quickly recovered. Thus, the target protein-enriched regions can be reliably achieved and identified at high-resolution mapping.
• Low Input Materials: Robust sonication-free fragmentation, unbound DNA cleavage, and immunocapture are all processed in the same single-tube with on-beads ligation. This method uses both cells and tissues and allows for maximal degradation protection of the target protein with minimal sample loss. As a result, the input cell amount can be as few as 500 cells, or chromatin amount can be as low as 0.1 µg.
• Minimal Background: Cleavage of unbound DNA sequences in the two (2) end of the target protein/DNA complex in situ enables the minimized immunocapture background, allowing the sequencing data analysis with <10 million reads.
• Fast, Streamlined Procedure: The procedure from cells to library DNA is less than 3 hours.
• Highly Convenient: The kit contains all required components for each step of the CUT&RUN, thereby allowing the EpiNext™ CUT&RUN Fast Kit to be the most convenient with reliable and consistent results. 

Background Information 
Enrichment of histone or transcription factor (TF)-complexed DNA in vivo followed by next-generation sequencing offers an advantageous tool for studying genome-wide protein-DNA interactions. It allows for the analysis of a specific protein binding to DNA sequences in living cells. Such analysis requires that the method reliably identify true target protein-enriched regions, particularly from limited cell samples. These samples could include rare cell populations isolated from tissues, specific cells sorted from entire cell populations, and primary cultured sub-population cells such as embryonic cells. In addition, the method should ensure that enriched DNA contains minimal background and that the mapping of protein-DNA binding regions is with minimal bias and at high resolution. A major method that has been used for a long time to achieve this goal is chromatin immunoprecipitation, followed by sequencing (ChIP-Seq). However, the main limitation for ChIP is that it needs 1) a large amount of input material, cells or tissue, to produce a strong enough signal over background noise; and 2) cross-linking during an initial fixation step. There are several advanced methods available for ChIP-Seq with reduced cell numbers or increased resolution. These methods include ChIP-exo and ChIPmentation. ChIP-exo provides high-resolution mapping, but is time-consuming and requires an ample supply of input cells. While ChIPmentation uses transposase and sequencing-compatible adaptors to enable the integration of ligation during the ChIP process, it follows a traditionally slow (2 days) ChIP procedure and cannot achieve high-resolution mapping. CUT&RUN (Cleavage Under Target & Release Using Nuclease) was developed to release the captured target protein/DNA complex from limited biological materials for mapping protein-DNA interaction, and it has significantly improved mapping resolution. However, CUT&RUN needs expensive PA/Mnase fusion protein that has significant A/T sequence bias, causing the target protein-interacted DNA region profiles to be seriously affected by the level of MNase digestion.   

Principle & Procedure 
The kit contains all necessary reagents required for carrying out a successful CUT&RUN starting from mammalian cells or isolated nuclei/chromatin. In the reaction, nuclei are isolated from cells. The target protein-DNA complex is bound/captured with the CHIP-grade antibody of interest. With the use of a unique nucleic acid cleavage enzyme mix, chromatin is fragmented, and DNA sequences in both ends of the target protein/DNA complex are cleaved/removed. At the same time, the DNA sequence occupied by the target protein is unaffected. The target protein-bound DNA is then purified and eluted. The enriched DNA can be used for the analysis of protein-DNA interaction with various methods, especially with next generation sequencing. Included in the kit are a positive control antibody (anti-H3K9me3), a negative control non-immune IgG, and control chromatin, which can be used to demonstrate the efficacy of the kit and performance at the PCR/bioanalyzer step. 

Starting Materials
Starting materials can include various mammalian cell samples such as culture cells from a flask or plate, primary cells, or rare cell populations isolated from blood, body fluid, fresh/frozen tissues (pre-prepared chromatin), and specific cells sorted from entire cell populations, and embryonic cells, etc.