Revision 9
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:

ChIP-seq, C&R

:

:

:

:

Product Information

Storage

Store all components at -20ºC. This product is stable for 12 months if stored properly.

Specificity / Sensitivity

This kit has been validated in combination with Multiplex Oligos for Illumina Systems (Single Index Primers) (ChIP-seq, CUT&RUN) #29580 or Multiplex Oligos for Illumina Systems (Dual Index Primers) (ChIP-seq, CUT&RUN) #47538 to generate qualified DNA libraries using as little as 0.5 ng ChIP DNA or as little as 0.1 ng CUT&RUN DNA as starting materials.

Species Reactivity:

All Species Expected

Product Description

Next generation sequencing (NG-seq) is a high throughput method that can be used downstream of chromatin immunoprecipitation (ChIP) and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) assays to identify and quantify target DNA enrichment across the entire genome. The DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) contains all of the enzymes and buffers necessary to generate high quality DNA sequencing libraries from ChIP or CUT&RUN DNA for next-generation sequencing on the Illumina Systems platform. The fast, user-friendly workflow minimizes hands-on time needed for generation and purification of DNA libraries. This product must be used in combination with Multiplex Oligos for Illumina Systems (Single Index Primers) (ChIP-seq, CUT&RUN) #29580 or Multiplex Oligos for Illumina Systems (Dual Index Primers) (ChIP-seq, CUT&RUN) #47538.

This product provides sufficient amounts of reagents for 24 reactions and is compatible with both enzymatic- or sonication-fragmented, ChIP-enriched DNA. Distinct protocols are provied for DNA library preparation from ChIP and CUT&RUN DNA. This product is compatible with SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003, SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005, SimpleChIP® Plus Sonication Chromatin IP Kit #56383, and CUT&RUN Assay Kit #86652. This product is not compatible with SimpleChIP® Enzymatic Chromatin IP Kit (Agarose Beads) #9002 and SimpleChIP® Plus Enzymatic Chromatin IP Kit (Agarose Beads) #9004 because agarose beads are blocked with sonicated salmon sperm DNA, which will contaminate DNA library preps and NG-seq.

Background

The chromatin immunoprecipitation (ChIP) assay is a powerful and versatile technique used for probing protein-DNA interactions within the natural chromatin context of the cell (1,2). This assay can be used to identify multiple proteins associated with a specific region of the genome, or the opposite, to identify the many regions of the genome bound by a particular protein (3-6). It can be used to determine the specific order of recruitment of various proteins to a gene promoter or to "measure" the relative amount of a particular histone modification across an entire gene locus (3,4). In addition to histone proteins, the ChIP assay can be used to analyze binding of transcription factors and co-factors, DNA replication factors and DNA repair proteins. When performing the ChIP assay, cells or tissues are first fixed with formaldehyde, a reversible protein-DNA cross-linking agent that "preserves" the protein-DNA interactions occurring in the cell (1,2). Cells are lysed and chromatin is harvested and fragmented using either sonication or enzymatic digestion. The chromatin is then immunoprecipitated with antibodies specific to a particular protein or histone modification. Any DNA sequences that are associated with the protein or histone modification of interest will co-precipitate as part of the cross-linked chromatin complex and the relative amount of that DNA sequence will be enriched by the immunoselection process. After immunoprecipitation, the protein-DNA cross-links are reversed and the DNA is purified. Standard PCR or Quantitative Real-Time PCR can be used to measure the amount of enrichment of a particular DNA sequence by a protein-specific immunoprecipitation (1,2). Alternatively, the ChIP assay can be combined with genomic tiling micro-array (ChIP on chip) techniques, high throughput sequencing, or cloning strategies, all of which allow for genome-wide analysis of protein-DNA interactions and histone modifications (5-8).

  1. Orlando, V. (2000) Trends Biochem Sci 25, 99-104.
  2. Kuo, M.H. and Allis, C.D. (1999) Methods 19, 425-33.
  3. Agalioti, T. et al. (2000) Cell 103, 667-78.
  4. Soutoglou, E. and Talianidis, I. (2002) Science 295, 1901-4.
  5. Mikkelsen, T.S. et al. (2007) Nature 448, 553-60.
  6. Lee, T.I. et al. (2006) Cell 125, 301-13.
  7. Weinmann, A.S. and Farnham, P.J. (2002) Methods 26, 37-47.
  8. Wells, J. and Farnham, P.J. (2002) Methods 26, 48-56.

Species Reactivity

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

Applications Key

ChIP-seq: Chromatin IP-seq C&R: CUT&RUN

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.
SimpleChIP is a registered trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.
Revision 9
#56795

DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN)

Chromatin Immunoprecipitation Image 1: DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) Expand Image
Figure 1. Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HCT 116 cells and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared from 50 ng, 5 ng, or 0.5 ng enriched ChIP DNA or 50 ng Input DNA using SimpleChIP® ChIP-seq Multiplex Oligos for Illumina (Dual Index Primers) #47538, pooled into one sample, and sequenced on an Illumina Next-Seq platform. The figure shows binding across GAPDH, a known target gene of H3K4me3. For additional ChIP-seq tracks, please download the product datasheet.
Chromatin Immunoprecipitation Image 2: DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) Expand Image
Figure 2. Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HCT 116 cells and TCF4/TCF7L2 (C48H11) Rabbit mAb #2569, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared from 50 ng, 5 ng, or 0.5 ng enriched ChIP DNA or 50 ng Input DNA using SimpleChIP® ChIP-seq Multiplex Oligos for Illumina (Dual Index Primers) #47538, pooled into one sample, and sequenced on an Illumina Next-Seq platform. The figure shows binding across CaMK2D, a known target gene of TCF4/TCF7L2. For additional ChIP-seq tracks, please download the product datasheet.
Chromatin Immunoprecipitation Image 3: DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) Expand Image
Figure 3. Metagene analysis of ChIP-seq data generated using different amounts of starting ChIP DNA. Analyses of both Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 (left) and TCF4/TCF7L2 (C48H11) Rabbit mAb #2569 (right) ChIP-seq data show that the signal-to-noise ratio for peaks identified across entire genome are similar for all three starting amounts of ChIP DNA.
Chromatin Immunoprecipitation Image 4: DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) Expand Image
Figure 4. Agilent Bioanalyzer System profiles of DNA libraries prepared from different starting amounts of ChIP DNA generated using the SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA libraries were prepared from 50 ng (A), 5 ng (B), and 0.5 ng (C) of enriched ChIP DNA and 50 ng (D) input DNA. As shown, each DNA library preparation shows a similar DNA fragment size (expected range 300 bp to 900 bp).
No image available
Product Image 1: DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) Expand Image
Figure 5. CUT&RUN NGS data generated using Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 and 3.6 ng of CUT&RUN DNA, as described in Table 2. The figure shows binding across chromosome 12 (upper), including GAPDH (lower), a known target gene of H3K4me3.
Product Image 2: DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) Expand Image
Figure 6. CUT&RUN NGS data generated using TCF4/TCF7L2 (C48H11) Rabbit mAb #2569 and 0.7 ng of CUT&RUN DNA, as described in Table 2. The figure shows binding across chromosome 8 (upper), including MYC (lower), a known target gene of TCF4/TCF7L2.
Product Image 3: DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) Expand Image
Figure 7. CUT&RUN NGS data generated using Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb #13499 and 3.1 ng of CUT&RUN DNA, as described in Table 2. The figure shows binding across chromosome 7 (upper), including ACTB (lower), a known target gene of Phospho-Rpb1.
Product Image 4: DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) Expand Image
Figure 8. Agilent Bioanalyzer System profiles of DNA libraries prepared from different starting amounts of CUT&RUN DNA generated using the Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751. DNA libraries were prepared from 1 ng (A), 0.5 ng (B), 0.3 ng (C), and 0.1 ng (D) of enriched CUT&RUN DNA. As shown, each DNA library preparation shows a similar DNA fragment size.
Image Gallery
Learn more about how we get our images
Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.