CUTANA™ pAG-MNase for ChIC/CUT&RUN Workflows - 250 Rxns

$1,250.00
SKU: 15-1116
Pack Size: 250 Rxns
Type:  Nuclease Host: E.coli
Mol Wgt: 44 kDa Epitope Tag: 6His


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

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

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

CUTANA™ pAG-MNase for ChIC/CUT&RUN Workflows Application Notes: This product is sufficient to perform 250 CUT&RUN reactions. Recommended use: 2.5 μL of the supplied enzyme into a 250 μL CUT&RUN reaction (20X dilution). For detailed applications and uses of this product, please see 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...”

Product References:
(1) Schmid M et al., Mol Cell. 2004; 16:147-157
(2) Skene PJ, Henikoff S. eLife 2017; 6:e21856
(3) Skene PJ et al., Nat Protoc. 2018; 13:1006-1019

View technical datasheet for this product. 15-1116 Datasheet
View CUTANA™ CUT&RUN Protocol for Histone PTMs and Transcription Factors. CUTANA™ CUT&RUN Protocol
View previous CUTANA™ CUT&RUN Protocol. CUTANA™ CUT&RUN Protocol
15-1016 Protein Gel Data

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.
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15-1016 Size Distribution of Released Chromatin

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™ II DNA 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).
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15-1016 CUT&RUN Data

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.
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