Chromatin immunoprecipitation (ChIP) is a popular tool for studying genome-wide protein/DNA interactions in cells, whereby target histone or transcription factor-complexed DNA is enriched, and the interaction regions are mapped, generally via next-generation sequencing. Due to the limitations of ChIP, mainly, 1) its requirement for large amounts of starting material, 2) long processing times, and 3) low resolution, Cleavage Under Target and Release Using Nuclease (CUT&RUN) emerged to address these issues. The technique, which is performed in situ on intact cells or nuclei without fixation, entails cleavage of chromatin at specific antibody-occupied sites by a pAG-MNase fusion protein and the subsequent release of protein/DNA complexes. CUT&RUN has several advantages over ChIP, including less starting material and higher resolution. However, the pAG-MNase fusion protein used in the procedure can generate non-specific cleavage by antibody un-coupled pAG-MNase, significantly limiting the specificity of CUT&RUN use for most transcription factors in different species and cell/tissue types and causing the target protein-interacted DNA region profiles to be seriously affected. In addition, CUT&RUN is still time-consuming.
CUT&RUN-Fast and CUT&LUNCH
An improved CUT&RUN technique, CUT&RUN-Fast, was developed by EpigenTek that employs a novel and 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, thereby increasing specificity and resolution. This also greatly speeds up the process and avoids overnight incubation.
Recently, EpigenTek has further refined the CUT&RUN-Fast method with the new Cleavage Under Target and Liberate Unique Nucleic Complex Homogenously (CUT&LUNCH), which features an extremely fast protocol, further enhanced specificity, and minimized background.
Performing a CUT&LUNCH Experiment
The CUT&LUNCH assay’s streamlined procedure allows you to selectively enrich target protein/DNA complexes very quickly. The rapid 3-step protocol can be completed in just under 2 hours, minimizing nuclear damage and chromatin loss while preserving native chromatin structure:
Step 1: Antibody Binding to Target (30 minutes)
Step 2: Enzyme Cleavage (10 minutes)
Step 3: Selective Recovery and Purification (60 minutes)
Advantages of CUT&LUNCH vs Traditional CUT&RUN and ChIP
CUT&LUNCH
Traditional CUT&RUN
ChIP
Workflow convenience
Convenient Only 19 steps total
Less convenient >50 steps total
Varies for different ChIP assays
Required cell amount
2,000 - 500,000
5,000 - 500,000
100 - 10,000,000
Profiled target range
Good for histones Good for TF proteins
Good for histone Poor for TF proteins
Good for histones Good for TF proteins
Specificity of cleaved and collected DNA
Stable; not dependent on quantity of target molecules contained in the sample
Not affected by target types and amounts
Unstable; varies depending on quantity of target molecules contained in the sample
Affected by target types and amounts
N/A
Sequence resolution
High
High
Medium
Negative/Positive Control Ratio (%)
5%
30%
20%
Assay time length
1 hour 50 minutes
6 hours - 2 days
Varies from 4 hours - 2 days for different ChIP assays
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