CUTANA™ pAG-MNase for ChIC/CUT&RUN Workflows

SKU: 15-1016
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  • Type:  Nuclease
  • Mol Wgt:  43.7 kDa
  • Host:  E. coli
  • Epitope Tag:  6xHis


Available in 50 and 250 Reaction pack sizes. CUTANA™ pAG-MNase is the key reagent for performing Chromatin Immunocleavage (ChIC) [1] and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) [2,3].

Add our Library Prep Kit for a streamlined CUT&RUN workflow

As a fusion of Proteins A and G to Micrococcal Nuclease, CUTANA pAG-MNase is compatible with target antibodies from a broad spectrum of host species and is highly purified to remove contaminating E. coli DNA, which can complicate analysis at low cell numbers. This enzyme enables efficient mapping of chromatin features in ChIC/CUT&RUN, allowing for significant improvements in signal to noise at reduced cell inputs and sequencing depth compared to ChIP-seq.

Validation Data

CUT&RUN was performed on 500k K562 cells with 0.5 µg of either IgG (EpiCypher 13-0042), H3K4me3 (EpiCypher 13-0041), or H3K27me3 (ThermoFisher MA5-11198) antibodies using CUTANA™ pAG-MNase (1:20 dilution) and the CUTANA™ ChIC/CUT&RUN Kit v3.0 (EpiCypher 14-1048). Library preparation was performed with 5 ng of DNA (or the total amount recovered if less than 5 ng) using the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001/14-1002). Both kit protocols were adapted for high throughput Tecan liquid handling. Libraries were run on an Illumina NextSeq2000 with paired-end sequencing (2x50 bp). Sample sequencing depth was 3.6 million reads (IgG), 4.3 million reads (H3K4me3), and 5.2 million reads (H3K27me3). Data were aligned to the hg19 genome using Bowtie2. Data were filtered to remove duplicates, multi-aligned reads, and blacklist regions.

Figure 1: CUT&RUN gene browser tracks
CUT&RUN was performed as described above. Data verifies low non-specific MNase digestion with the absence of peaks in the IgG track, an expected H3K4me3 profile with sharp promoter peaks, and broad peaks in heterochromatin regions consistent with H3K27me3. Image was generated using the Integrative Genomics Viewer (IGV, Broad Institute).

Figure 2: Size distribution of released chromatin
CUT&RUN was performed as described above. Excised DNA is highly enriched for mononucleosomes (peaks at ~300 bp represent 150 bp nucleosomes + sequencing adapters).

Figure 3: CUT&RUN genome-wide heatmaps
CUT&RUN was performed as described above. Heatmaps show CUT&RUN signal aligned to annotated transcription start sites (TSS, +/- 2kb). High and low signal are ranked by intensity (top to bottom) and colored such that red indicates high localized enrichment and blue denotes background signal. Gene rows in each heatmap are aligned and sorted from high to low signal relative to H3K4me3 (middle).

Figure 4: Protein gel data
CUTANA™ pAG-MNase (1 µg) was resolved via SDS-PAGE and stained with Coomassie blue. The migration and molecular weight of the protein standards are indicated.

Recommended Accessory Reagents

Item CAT
CUTANA™ CUT&RUN Library Prep Kit 14-1001
CUTANA™ DNA Purification Kit 14-0050
CUTANA™ Concanavalin A Conjugated Paramagnetic Beads 21-1401
SNAP-CUTANA™ K-MetStat Panel 19-1002
CUT&RUN Antibodies See the list
CUTANA™ E. coli Spike-in DNA 18-1401
Magnetic Separation Rack, 0.2 mL Tubes 10-0008
Magnetic Separation Rack, 1.5 mL Tubes 10-0012
CUTANA™ CUT&RUN 8-strip 0.2 mL Tubes 10-0009

Technical Information

Stable for one year at -20°C from date of receipt. The protein is not subject to freeze/thaw under these conditions.
10 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, and 50% glycerol.

Application Notes

Add 2.5 µL of the supplied enzyme to a 50 µL CUT&RUN reaction (20X dilution). For detailed applications and uses of this product, please see our CUT&RUN protocol.


Background References:
[1] Schmid et al. Mol. Cell. (2004). PMID: 15469830
[2] Skene & Henikoff eLife (2017). PMID: 28079019
[3] Skene et al. Nat. Protoc. (2018). PMID: 29651053

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