|Description||ChIC/CUT&RUN Kit (pA-MN)|
|Background Information||Protein-DNA interactions are essential to almost all biological processes, such as DNA replication and repair, transcription and regulation of gene expression. Among many techniques interrogating protein-DNA interactions, chromatin immunoprecipitation (ChIP) has been the dominant method. Even though improvements have been made to its readout strategies, ChIP has many problems and inefficiencies, such as high background that limits sensitivity, high amounts of cellular input, and artifacts resulting from cross-linking and protein solubilization.
In 2004, from the Laemmli lab, Schmid et al. reported a novel alternative to ChIP to detect binding sites of transcription factors in the genome by targeting micrococcal nuclease (MNase) conjugated with protein A (pA-MN) to specifically cleave DNA at regions interacting with a protein of interest through a specific antibody. Importantly, MNase is activated and cleaves only in the presence of Ca2+ ions, thus allowing for controlled DNA cleavage. This novel method is termed chromatin immuno-cleavage (ChIC)1. As a result, protein-DNA mapping resolution was improved to 10-200bp with excellent specificity. Furthermore, in 2018, Skene et al. in the Henikoff lab combined ChIC with sequencing to detect genome-wide transcription factor binding sites and histone modifications on native chromatin and termed Cleavage Under Targets and Release Using Nuclease (CUT&RUN)2.
In the ChIC/CUT&RUN methods, only the targeted fragments are released into solution and the majority of DNA is left behind, leading to an exceptionally low background level. As a result, ChIC/CUT&RUN provides high-quality results using only 100 cells for a histone modification analysis and 1,000 cells for a transcription factor binding site analysis. In contrast to ChIP, ChIC/CUT&RUN is free of solubility issues and DNA accessibility artifacts from cross-linking solubilization. ChIC/CUT&RUN out-performs ChIP in resolution, signal-to-noise ratio, and depth of sequencing required.
Based on these two publications, we developed ChIC/CUT&RUN kits to enable researchers conveniently perform ChIC/CUT&RUN experiments. The kits supply all key reagents and buffers to prepare ChIC/CUT&RUN samples before the DNA sequencing library preparation step. There are three kit configurations namely with protein A-micrococcal nuclease (pA-MN, Cat. No. CHR100), protein G-micrococcal nuclease (pG-MN, Cat. No. CHR101) and pA-MN/pG-MN fusion protein blend (Cat. No. CHR102). pA-MN has higher affinity to most antibodies including rabbit IgG, whereas pG-MN is more suitable for antibodies that have low affinity to protein A for example mouse IgG1. pA-MN/pG-MN blend allows for either choice. MilliporeSigma also supply DNA extraction kit for ChIC/CUT&RUN (12 rxns, Cat. No. CHR104), and 2% digitonin solution (Cat. No. CHR103) as accessory products for ChIC/CUT&RUN assays.
In additiona, a ChIC and CUT&RUN data analysis tool is created to enable our customers to process and analyze the resulting data yourself. For customers who purchased our ChIC/CUT&RUN kits, they can access the data analysis tool for free by following the steps in the instruction sheet found in the ChIC/CUT&RUN kit’s package. Your order ID can be found on the packing slip, the order confirmation or retrieved by calling your local Customer Service. For more information about the data analysis tool, please view here.
1. Schmid, M., Durussel,T., Laemmli, U.K. ChIC and ChEC: genomic mapping of chromatin proteins. Molecular Cell. 16, 147-157 (2004).
2. Skene, P. J., Henikoff J. G., Henikoff, S. Targeted in situ genome-wide profiling with high efficiency for low cell numbers. Nature Protocols. 13, 1006-1019 (2018).
|Application||A novel alternative to ChIP with a much faster procedure,extremely low signal-to-noise ratio and much less starting sample.|
|Application Notes||This kit provides the following benefits for your genome-wide profiling of chromatin protein interactions.
• Extremely low signal-to-noise ratio than cross-linking ChIP-seq (Because of the very low background with ChIC/CUT&RUN, typically 5 million paired-end reads per sample suffice for TFs or nucleosome modifications).
• Cross-linking eliminated thus sonicators or high-speed spin steps not needed.
• Much lower number of starting cells (minimum 100 cell for histone modification and 1,000 cells for a transcription factor).
• Much shortened procedure time – from cells to purified DNA completed within one day (minimum 3 hours).
• No special equipment needed, using standard equipment that is already present in most molecular biology laboratories.
For more information, please view here.
|Concentration||Please refer to lot specific datasheet.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Store ChIC component box 1 at 2°C-8ºC and ChIC component box 2 at -20°C; performance guaranteed for 6 months from date of receipt when reagents are stored properly.|
|Material Size||12 reactions|
|ChIC (Chromatin Immuno-Cleavage) Kits|