CUTANA™

CUTANA™ pAG-MNase for ChIC/CUT&RUN Workflows - 50 rxns

$300.00
SKU: 15-1016
Pack Size: 50 Reactions
  • Type:  Nuclease
  • Mol Wgt:  43.7 kDa
  • Host:  E. coli
  • Epitope Tag:  6xHis

Description

Recombinantly produced in E. coli, CUTANA pAG-MNase for ChIC/CUT&RUN Workflows is a fusion of Proteins A and G to Micrococcal Nuclease. This construct is useful in performing Chromatin Immunocleavage (ChIC) (1) and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) (2, 3). CUTANA pAG-MNase contains a C-terminal 6xHis epitope tag.

Validation Data

Figure 1: 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.

Figure 2: Size Distribution of Released Chromatin
CUT&RUN was performed using CUTANA pAG-MNase (1:20 dilution) with 0.5 million K-562 cells. Purified DNA was subjected to sequencing library preparation using an NEBNext® Ultra™ DNA II Library Prep Kit for Illumina®. Agilent Bioanalyzer traces for libraries derived from H3K4me3 CUT&RUN (blue track; ThermoFisher Scientific Catalog No. PA5-27029, Lot SG2419844A) and H3K27me3 CUT&RUN (orange track; Cell Signaling Technology Catalog No. 9733, Lot 14) are shown. Excised DNA is highly enriched for mononucleosomes (peak at 300 bp reflects 150 bp insert size).

Figure 3: CUT&RUN Data
Sequencing tracks obtained using CUTANA pAG-MNase. (A) CUT&RUN was performed using 0.5 million K-562 cells with H3K4me3 antibody (ThermoFisher Scientific Catalog No. PA5-27029, Lot SG2419844A). Reference ChIP-seq tracks from ENCODE using 20 million cells (GEO Accession GSM2534288, Run SRR5339104 at locus chr1:26,732,009-26,900,000) are shown for comparison. (B) CUT&RUN was performed using 0.5 million K-562 cells with H3K27me3 antibody (Cell Signaling Technology Catalog No. 9733, Lot 14). Reference ChIP-seq tracks from ENCODE using 20 million cells (GEO Accession GSM733658, combined Runs SRR227389 + SRR227390 at locus chr20:35,914,690-36,600,000) are shown for comparison. Total read depth for each experiment prior to alignment is indicated on the browser tracks. In both cases, CUTANA pAG-MNase achieved superior sensitivity and signal-to-noise with >10-fold reduced sequencing depth and 40-fold less cell input.

For more information regarding the ChIP-seq methods and ENCODE resource, see Ram et al., Cell 2011; 147:1628-1639 and Nature 2012; 489:57-74.

Technical Information

Formulation
CUTANA pAG-MNase is provided as a 20X stock in 10 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, and 50% glycerol.
Storage and Stability
Stable for one year at -20°C from date of receipt. The protein is not subject to freeze/thaw under these conditions.

Application Notes

This product is sufficient to perform 50 CUT&RUN reactions.

Recommended Use: 2.5 µL of the supplied enzyme into a 50 µL CUT&RUN reaction (20X dilution). For detailed applications and uses of this product, please see our CUT&RUN protocol or Skene et al., 2018 (3).

Application Notes Addendum: Since CUT&RUN has lower background and is compatible with fewer cells compared to ChIP-seq, it is not recommended to assess fragment size distribution using agarose gel or capillary electrophoresis (e.g. Agilent Bioanalyzer or TapeStation) prior to library preparation. This analysis is not indicative of the success of a CUT&RUN experiment, and further the amount of DNA recovered may be below the sensitivity of detection for these approaches. To gauge the success of a CUT&RUN experiment, assess DNA yield compared to + and - IgG controls, determine fragment size distribution of sequence-ready libraries, and evaluate peak structure and expected genome-wide distribution in NGS data. As per Skene et al. (2018), page 1014, step 37: “targeted DNA recovered is too low in amount and too small in size to be detected by gel analysis...” For more detailed information, please see our CUT&RUN protocol.

References

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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