CUT&RUN (Cleavage Under Targets & Release Using Nuclease)은 in vivo에서 표적 특이적 1차 항체와 Protein A-Protein G-Micrococcal Nuclease (pAG-MNase)를 이용하여 특정 protein-DNA 복합체를 분리하는 방법입니다. 기존 ChIP (chromatin-immunoprecipitation) 및 ChIP-seq에 비해 효율적으로 chromatin profiling을 가능하게 합니다.
HOW CUT&RUN Works
Ab & pAG-MNase binding:
1. Cells are immobilized on Concanavalin A Magnetic Beads to allow for subsequent buffer and reagent exchanges.
2. Cell membranes are then permeabilized with digitonin to facilitate the entry of primary antibody and pAGMNase fusion enzyme into the cell nuclei.
3. The target-specific primary antibody recruits the pAG-MNase to the chromatin through protein-protein interactions between the antibody and the pAG domain of the fusion enzyme.
4. The addition of Ca2+ activates the pAG-MNase, which gently cleaves and liberates the desired chromatin fragments, allowing them to diffuse away from the genomic chromatin, out of the cell, and into the supernatant.
5. DNA is purified using DNA purification spin columns or phenol/chloroform extraction followed by ethanol precipitation. The purified, enriched DNA is then identified and quantified using qPCR or NG-seq.
The Benefits of CUT&RUN
|Low sample requirement||Only 100K cells needed|
|Fast time to results||1-2 days from cell to DNA|
|Sequencing cost savings||Only 3-5 million high-quality reads required|
|Target versatility||Generate sequencing and/or qPCR data for histones, histone modifications, transcription factors, and cofactors|
|Antibody versatility||Compatible with rabbit and mouse antibodies|
|Reproducible results||Spike-In control DNA to normalize signal between samples|
|Avoid “cross linking” artifacts||An in vivo method performed using native chromatin|
The CUT&RUN Assay Kit from CST only requires 100,000 cells and works as well as ChIP-seq
CUT&RUN and ChIP assays were performed with HCT 116 cells (1×105 for CUT&RUN, 4×106 cells for ChIP) and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751, using the CUT&RUN Assay Kit #86652 or the SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. Comparison of enrichment at the GAPDH gene, a known target of H3K4me3. The input track is from the CUT&RUN input sample.
CUT&RUN Kits and Reagents
Cell Signaling Technology에서는 엄격한 in-house validation을 거친 CUT&RUN 연구를 위한 제품을 제공합니다. 누구나 쉽게 분석이 가능하도록 자세한 프로토콜과 함께 실험에 필요한 모든 버퍼와 시약이 포함된 CUT&RUN 키트와 다양한 관련 제품을 만나보세요.
|86652||CUT&RUN Assay Kit|
|40366||CUT&RUN pAG-MNase and Spike-in DNA|
|14209||DNA Purification Buffers and Spin Columns (ChIP, CUT&RUN)|
|88989||SimpleChIP® Universal qPCR Master Mix|
|56795||SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina®|
|47538||SimpleChIP® ChIP-seq Multiplex Oligos for Illumina® (Dual Index Primers)|
|29580||SimpleChIP® ChIP-seq Multiplex Oligos for Illumina® (Single Index Primers)|
|66362||Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN)|
|93569||Concanavalin A Magnetic Beads and Activation Buffer|
|7012||Protease Inhibitor Cocktail (200X)|
|7013||RNAse A (10 mg/ml)|
|10012||Proteinase K (20mg/ml)|
|31415||CUT&RUN 10X Wash Buffer|
|15338||CUT&RUN Antibody Binding Buffer|
|48105||CUT&RUN 4X Stop Buffer|
|42015||CUT&RUN DNA Extraction Buffer|