Revision 2

#77552Store at RT, 4C, -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

C&T

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Product Information

Storage

All components in this kit are stable for 6 months when stored at the recommended temperature.

Specificity / Sensitivity

The CUT&Tag Assay Kit can be utilized with any CUT&Tag-validated antibody to detect endogenous levels of protein-DNA interactions and histone modifications in mammalian cells (see Figures 1–3). One CUT&Tag reaction can use between 5,000 to 250,000 cells or 1 to 5 mg of tissue of starting material (see Figures 5-7). The kit is compatible with multiple species of antibodies, including rabbit and mouse (see Figure 4). The positive control Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 detects multiple species of tri-methyl histone H3 Lys4 protein, including human, mouse, rat, and monkey.

Product Description

The CUT&Tag Assay Kit is designed to conveniently provide reagents needed to perform up to 24 reactions. The kit has been optimized to work in fresh or lightly fixed cells for all types of DNA-binding proteins, including histones, transcription factors, and cofactors. In addition, the kit has been optimized to work for histones in fresh or lightly fixed tissues. For analysis of transcription factors and cofactors in tissues, we recommend using the CUT&RUN Assay Kit #86652. If possible, we recommend using 100,000 cells or 1 mg of tissue per CUT&Tag reaction. If starting cell number is limited, this kit is validated to work with as few as 5,000 to 10,000 cells per reaction for histone modification targets and as few as 20,000 cells per reaction for transcription factors and cofactors. This kit is compatible with both whole cells and nuclei as starting material. We have not found that using nuclei generates a stronger signal or a higher signal-to-noise reaction compared to whole cells. A complete assay can be performed in as little as one day. The CUT&Tag Assay Kit also provides important controls to ensure a successful CUT&Tag experiment, including positive control Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 and negative controls Normal Rabbit IgG #2729 and Normal Mouse IgG #68860. The negative control Normal Rabbit IgG #2729 or Normal Mouse IgG #68860 is optional for the experiments, depending on the requirements of the peak calling algorithm used. This kit is compatible with downstream Next Generation sequencing (NG-seq) analysis, but not qPCR.

Background

Similar to Cleavage Under Targets and Release Using Nuclease (CUT&RUN), Cleavage Under Targets and Tagmentation (CUT&Tag) is a powerful technique used for probing protein-DNA interactions within the natural chromatin context of the cell (1-3). CUT&Tag has many of the same advantages as the CUT&RUN assay in that it provides a rapid, robust, and true low cell number protocol for detection of protein-DNA interactions in the cell. In addition, the CUT&Tag assay adds an in situ adaptor DNA ligation step carried out by the pAG-Tn5 enzyme, in which an adaptor DNA is ligated directly to antibody-targeted chromatin DNA fragments in the cell. As a result, subsequent DNA library preparation is much faster and easier than library preparation following the CUT&RUN assay, free from DNA end repair, A-tailing, and adaptor ligation in vitro. CUT&Tag works very well for analyzing histone modifications, in addition to mapping some transcription factor and cofactor binding.

  1. Kaya-Okur, H.S. et al. (2019) Nat Commun 10, 1930.
  2. Kaya-Okur, H.S. et al. (2020) Nat Protoc 15, 3264-3283.
  3. Henikoff, S. et al. (2021) Bio Protoc 11, e4043.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Applications Key

C&T: CUT&Tag

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
CUT&Tag provided under a license from Active Motif, Inc. under U.S. Patent No. 10,689,643 and 9,938,524, foreign equivalents, and child patents deriving therefrom. For purchaser's internal research use only. May not be used for resale, services, or other commercial use.
U.S. Patent No. 11,733,248, foreign equivalents, and child patents deriving therefrom.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

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Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
仅供研究使用。不得用于诊断流程。
Revision 2
#77552

CUT&Tag Assay Kit

CUT and Tag Image 1: CUT&Tag Assay Kit Expand Image
图 1. CUT&Tag, CUT&RUN, and ChIP-seq assays were performed with NCCIT cells and Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb #9733, using this CUT&Tag Assay Kit, the CUT&RUN Assay Kit #86652, or the SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA libraries were prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415 for CUT&Tag samples and DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795 for ChIP-seq and CUT&RUN samples. The upper panel compares enrichment around HoxA genes, while the lower panel compares enrichment around HoxD genes, both are known target genes of H3K27me3.
CUT and Tag Image 2: CUT&Tag Assay Kit Expand Image
图 2. CUT&Tag, CUT&RUN, and ChIP-seq assays were performed with MCF7 cells grown in phenol red free medium and 5% charcoal stripped FBS for 4 d, then treated with β-estradiol (10 nM) for 45 min and Estrogen Receptor α (D8H8) Rabbit mAb #8644, using this CUT&Tag Assay Kit, the CUT&RUN Assay Kit #86652, or the SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA libraries were prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415 for CUT&Tag samples and DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795 for ChIP-seq and CUT&RUN samples. The upper panel compares enrichment of Estrogen Receptor α across chromosome 21, while the lower panel compares enrichment around TFF1, a known target gene of Estrogen Receptor α.
CUT and Tag Image 3: CUT&Tag Assay Kit Expand Image
图 3. CUT&Tag, CUT&RUN, and ChIP-seq assays were performed with NCCIT cells and JARID2 (D6M9X) Rabbit mAb #13594, using this CUT&Tag Assay Kit, the CUT&RUN Assay Kit #86652, or the SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA libraries were prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415 for CUT&Tag samples and DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795 for ChIP-seq and CUT&RUN samples. The upper panel compares enrichment around HoxA genes, while the lower panel compares enrichment around HoxD genes, both are known target genes of JARID2.
CUT and Tag Image 4: CUT&Tag Assay Kit Expand Image
图 4. CUT&Tag assay was performed with HeLa cells and either Rpb1 CTD (4H8) Mouse mAb #2629 or Phospho-Rpb1 CTD (Ser2/Ser5) (D1G3K) Rabbit mAb #13546, using this CUT&Tag Assay Kit. 使用 CUT&Tag Dual Index Primers 和 PCR Master Mix for Illumina Systems #47415 制备 DNA 文库。The upper panel compares enrichment of Rpb1 across chromosome 12, while the lower panel compares enrichment around GAPDH, a known target gene of Rpb1.
CUT and Tag Image 5: CUT&Tag Assay Kit Expand Image
图 5. CUT&Tag assay was performed with 100,000, 20,000, or 5,000 NCCIT cells (as indicated) and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751, using this CUT&Tag Assay Kit. 使用 CUT&Tag Dual Index Primers 和 PCR Master Mix for Illumina Systems #47415 制备 DNA 文库。上小图比较了遍及第 12 号染色体的 H3K4me3 富集，而下小图比较了 H3K4me3 的已知靶标基因 GAPDH 周围的富集。
CUT and Tag Image 6: CUT&Tag Assay Kit Expand Image
图 6. CUT&Tag assay was performed with 100,000 or 20,000 Hep G2 cells treated with Thapsigargin #12758 (300 nM) for 4h (as indicated) and ATF-4 (D4B8) Rabbit mAb #11815, using this CUT&Tag Assay Kit. 使用 CUT&Tag Dual Index Primers 和 PCR Master Mix for Illumina Systems #47415 制备 DNA 文库。上小图比较了遍及第 12 号染色体的 ATF-4 富集，而下图则比较了 ATF-4 的已知靶标基因 DDIT3/CHOP 周围的富集。
CUT and Tag Image 7: CUT&Tag Assay Kit Expand Image
图 7. CUT&Tag assay was performed with 1 mg of fresh mouse brain, heart, or liver tissues (as indicated) and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751, using this CUT&Tag Assay Kit. 使用 CUT&Tag Dual Index Primers 和 PCR Master Mix for Illumina Systems #47415 制备 DNA 文库。The figure shows enrichment of H3K4me3 around its known target gene GAPDH.
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Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
仅供研究使用。不得用于诊断流程。