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. 업그레이드 된 Cell Signaling Technology의 protocol로 더 빠르고 높은 효율의 CUT&RUN을 경험하세요!
CUT&RUN Assay Kit #86652의 “Mini” size 출시!
CUT&RUN를 경험해 보고 싶지만 24 assay가 가능한 기존 CUT&RUN Assay Kit #86652S 제품이
부담스러우셨던 분들을 위해 8번의 assay가 가능한 “Mini” kit가 출시 되었습니다.
Mini kit를 통해 Cell Signaling Technology의 CUT&RUN을 보다 쉽게 만나보세요.
• #86652P CUT&RUN Assay Kit (8 Assays)
• #86652S CUT&RUN Assay Kit (24 Assays)
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.
MNase Digestion:
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.
DNA Purification:
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.
Expended Benefits of CST CUT&RUN Assay Kit
| Low sample requirement | 5-10K cells for Histones 10-20K cells for Transcription Factors and Cofactors |
| Various sample types available |
Now compatible with fixed cells, fixed or fresh tissue and primary cells |
| 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 |
Even Lower Sample Requirement
CUT&RUN was performed with decreasing numbers of cells using 10,000, or 5,000 HeLa cells (as indicated) and Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb #9733.
DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figures show binding across chromosome 20 (upper), including
MYT1 (lower), a known target gene of H3K27me3+.
Validated for Use with Tissues
CUT&RUN was performed with 2.5 mg of medium-fixed mouse liver tissue (0.1% formaldehyde, 10 minutes) and SUZ12 (D39F6) XP® Rabbit mAb #3737 or RING1B (D22F2) XP® Rabbit mAb #5694, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for
Illumina® #56795. The figure shows binding of SUZ12 and RING1B across HoxA gene region.
Compatible for Use with Primary Cells
CUT&RUN was performed with 100,000 live human CD8+ T cells and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 or CTCF (D31H2) XP® Rabbit mAb #3418, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using SimpleChIP® ChIP-seq
DNA Library Prep Kit for Illumina® #56795. The figure shows binding across chromosome 8.
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.
| Catalog # | Product |
|---|---|
| 86652P | CUT&RUN Assay Kit (8 Assays) 신제품 |
| 86652S | CUT&RUN Assay Kit (24 Assays) |
| 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 |
| 16359 | Digitonin Solution |
| 27287 | 100X Spermidine |
| 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 |
