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CST CUT&RUN Kit: Validated for Flexibility

Same Kit, More Flexibility

The Cleavage Under Targets & Release Using Nuclease (CUT&RUN) assay is a faster, more cost-effective alternative to ChIP-qPCR and ChIP-seq that requires fewer cells to investigate protein-DNA interactions. Cell Signaling Technology® (CST®) has made its CUT&RUN Kit even better by performing additional validation and optimization to improve the protocol, resulting in even more options for how you explore protein-DNA interactions.

CUT&RUN

Expanded Benefits

CUT&RUN is an in vivo method that delivers results in 1-2 days, lowers sequencing costs, and is compatible with a variety of antibodies and targets. The CUT&RUN Assay Kit now offers these additional benefits to increase assay flexibility:

Improved protocols Optimized protocols enhance the enrichment of low abundance and/or weak binding transcription factors and cofactors
Even lower sample requirement 5-10K cells for Histones
10-20K cells for Transcription Factors and Cofactors
Now compatible with fixed cells Light cell fixation keeps cells intact, preserves cell signaling pathways, and enhances the enrichment for accessory components of huge complexes
Use fixed or fresh tissue samples with confidence Validated and optimized tissue protocols give you data confidence when using ~20x less sample compared to ChIP
Study protein-DNA interactions in primary cells CUT&RUN significantly lowers the cell number requirements, making it suitable to use with primary cells

Plus, don’t forget that the CUT&RUN Assay Kit from CST provides:

短時間でデータを取得可能 1-2 days from cell to DNA
Lower sequencing depth = lower sequencing costs Only requires 3-5 million high-quality reads per sample due to the inherently low assay background
In Vivo Method Assays are performed using native chromatin, eliminating cross-linking artifacts
多様な抗体に対応 ラビット抗体およびマウス抗体をご利用いただけます
多様な標的に対応 Generate sequencing and/or qPCR data for histones, histone modifications, transcription factors, and cofactors.
Straightforward quantification Spike in control DNA to simplify data quantification and normalization.

Better Data with an Improved Protocol

Continued development on the CUT&RUN protocol has further optimized the protocol to enhance your data and validated the CUT&RUN Assay Kit for use with a wider variety of sample types. For example, we now recommend performing digestions at 4°C instead of 0°C to facilitate the recovery of targeted chromatin fragments without significantly increasing background. This improves qPCR and NG-seq analysis, especially for transcription factors and cofactors.

Panel A

4°C vs. 0°C CUT&RUN qPCR data SUZ12

Panel B

4°C vs. 0°C CUT&RUN NGS data SUZ12

CUT&RUN was performed with NCCIT cells and SUZ12 (D39F6) XP® Rabbit mAb #3737, using CUT&RUN Assay Kit #86652. DNA was digested at either 0°C or 4°C as indicated. In panel A, the enriched DNA was quantified by real-time PCR using SimpleChIP® Human HoxA1 Intron 1 Primers #7707, SimpleChIP® Human HoxA2 Promoter Primers #5517, and SimpleChIP® Human α Satellite Repeat Primers #4486. 各サンプルの免疫沈降されたDNAの量を、Inputクロマチンの総量 (1に相当) に対する相対量で示しました。In panel B, DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across HoxA genes.

 

Save Time by Using 5-20x Less Sample

CUT&RUN already required significantly fewer cells to generate reliable data compared to ChIP-qPCR and ChIP-seq, and CST has further increased your time savings by optimizing protocols to use even less sample.

Unable to collect 100K cells, but still want to perform CUT&RUN? Not a problem as you can generate the same great data with just 5-20K cells.

Histones

Similar binding patterns were observed when 100,000, 10,000, or 5,000 cells were used on the CUT&RUN Assay Kit #86652 when studying histones.

Panel A

H3K4me3 NGS data for 100K, 10K & 5K cells

Panel B

H3K27me3 NGS data for 100K, 10K & 5K cells

CUT&RUN was performed with decreasing numbers of cells using CUT&RUN Assay Kit #86652. In panel A, 100,000, 10,000, or 5,000 HCT 116 cells and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751. DNAライブラリーは、SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795を用いて調製しました。図は、H3K4me3の既知の標的遺伝子であるGAPDH (下パネル) および、これを含む12番染色体全体への結合 (上パネル) を示しています。In panel B, CUT&RUN was performed with 100,000, 10,000, or 5,000 HeLa cells (as indicated) and Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb #9733. DNAライブラリーは、SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795を用いて調製しました。The figures show binding across chromosome 20 (upper), including MYT1 (lower), a known target gene of H3K27me3.

転写因子

Similar binding patterns were observed when 100,000, 20,000, or 10,000 cells were used on the CUT&RUN Assay Kit #86652 when studying transcription factors.

Panel A

Phospho-c-Jun NGS data for 100K, 20K & 10K cells

Panel B

TCF4 NGS data for 100K, 20K & 10K cells

CUT&RUN was performed with decreasing numbers of cells using CUT&RUN Assay Kit #86652. In panel A, CUT&RUN was performed with 100,000, 20,000, or 10,000 PC-12 starved overnight and treated with Human β-Nerve Growth Factor (hβ-NGF) #5221 (50 ng/mL) for 2 hrs and Phospho-c-Jun (Ser73) (D47G9) XP® Rabbit mAb #3270. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across chromosome 2 (upper), including Dclk1 (lower), a known target gene of Phospho-c-Jun. In panel B, CUT&RUN was performed with 100,000, 20,000, or 10,000 HCT 116 cells and TCF4/TCF7L2 (C48H11) Rabbit mAb #2569. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figures show binding across chromosome 8 (upper), including MYC (lower), a known target gene of TCF4.

コファクター

Similar binding patterns were observed when 100,000, 20,000, or 10,000 cells were used on the CUT&RUN Assay Kit #86652 when studying cofactors.

SUZ12 NGS data for 100K, 20K & 10K cells

CUT&RUN was performed with 100,000, 20,000, or 10,000 NCCIT cells and SUZ12 (D39F6) XP® Rabbit mAb #3737, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figures show binding across HoxD (upper) and HoxA (lower), a known target gene of SUZ12.

 

Study Protein-DNA Interactions in Tissue Samples with Confidence

The high sample input required to perform ChIP experiments is extremely restrictive when it comes to profiling chromatin in limited sample types like tissue. The sample savings CUT&RUN delivers for cultured cells extends to tissue samples. Depending on the tissue, ChIP can require anywhere from 25 to 50 mg of tissue while our CUT&RUN kit now only requires approximately 1 to 2.5 mg depending on the tissue type.

Validated protocols give you confidence in your data even when using ~20x less sample to explore protein-DNA interactions using the CST CUT&RUN Assay Kit #86652.

Heart Tissue – Histones and Transcription Factors

Only 1 mg of heart tissue was required to analyze protein-DNA interactions for H3K4me3 and transcription factor MITF using our CUT&RUN Kit.

H3K4me3 and MITF NGS data for 1mg Heart Tissue

CUT&RUN was performed with 1 mg of lightly fixed mouse heart tissue (0.1% formaldehyde, 2 minutes) and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 or MITF (D3B4T) Rabbit mAb #97800, using CUT&RUN Assay Kit #86652. DNAライブラリーは、SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795を用いて調製しました。The figure shows binding of H3K4me3 and MITF across chromosome 19 (upper), including Ifit2 gene (lower).

Brain Tissue – Histones and Transcription Factors

Only 1 mg of brain tissue was required to analyze protein-DNA interactions for H3K4me3 and transcription factor Brn2/POU3F2 using our CUT&RUN Kit.

H3K4me3 and Brn2 NGS data for 1 mg Brain Tissue

CUT&RUN was performed with 1 mg of lightly fixed mouse brain tissue (0.1% formaldehyde, 2 minutes) and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 or Brn2/POU3F2 (D2C1L) Rabbit mAb #12137, using CUT&RUN Assay Kit #86652. DNAライブラリーは、SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795を用いて調製しました。The figure shows binding of H3K4me3 and Brn2 across chromosome 6 (upper), including Snrpg gene (lower).

Liver Tissue – Histones and Transcription Factors

Only 1 mg of liver tissue was required to analyze protein-DNA interactions for H3K4me3 and the Glucocorticoid Receptor transcription factor using our CUT&RUN Kit.

H3K4me3 and Glucocorticoid Receptor NGS data for 1mg Liver Tissue

CUT&RUN was performed with only 1 mg of medium-fixed mouse liver tissue (0.1% formaldehyde, 10 minutes) and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 or Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb #12041, using CUT&RUN Assay Kit CUT&RUN Assay Kit #86652. DNAライブラリーは、SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795を用いて調製しました。The figure shows binding of H3K4me3 and Glucocorticoid Receptor across chromosome 8 (upper), including Mt2 gene (lower).

Liver Tissue – Cofactors

Only 2.5 mg of liver tissue were required to analyze protein-DNA interactions for the cofactors SUZ12 and RING1B using our CUT&RUN Kit.

SUZ12 and RING1B NGS data for 2.5mg Liver Tissue

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ライブラリーは、SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795を用いて調製しました。The figure shows binding of SUZ12 and RING1B across HoxA (upper) and HoxD (lower) gene regions.

 

Profile Chromatin in Primary Cells

Before CUT&RUN, analyzing protein-DNA interactions in primary cells, which aren’t immortalized, with ChIP was nearly impossible due to the challenges involved with trying to grow up enough cells.

The CUT&RUN Assay Kit #86652 significantly lowers the cell number requirement, making studying protein-DNA interactions in primary cells feasible. Don’t compromise your desired sample type just to meet cell number requirements.

H3K4me3 and CTCF data for CD8+ T 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 upper panel shows binding across chromosome 8, while the lower panel shows enrichment around the MYC gene, a known target of H3K4me3 and CTCF. See additional figure containing CUT&RUN-qPCR data in #3418 datasheet.

 

Product Options

CST offers CUT&RUN reagents in a kit or a la carte, so you can order one kit to get all the necessary reagents or order just the reagents you need. They are all validated in-house to ensure you’ll get the high-quality reagents you’ve come to expect from CST.

製品番号 製品
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
16359 Digitonin Solution
27287 100X Spermidine
7012 Protease Inhibitor Cocktail (200X)
7013 RNAse A (10 mg/mL)
10012 Proteinase K (20 mg/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
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