CUTANA™ pAG-MNase for ChIC/CUT&RUN Workflows · 2019-08-12 · Catalog No. 15-1016 Lot No....
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15-1016 Catalog No. 19199003 Lot No. Product Descrip5on: Recombinantly produced in E. coli, CUTANA TM pAG- MNase for ChIC/CUT&RUN Workflows is a fusion of Proteins A and G to Micrococcal Nuclease. This construct is useful in performing ChromaNn Immunocleavage (ChIC) (1) and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) (2,3). CUTANA TM pAG-MNase contains a C-terminal 6xHis epitope tag. 50 reacNons Pack Size 150kDa 100kDa 75kDa 50kDa 37kDa 25kDa 20kDa 15kDa Protein Gel Data: CUTANA TM pAG-MNase (1 µg) was resolved via SDS-PAGE and stained with Coomassie blue. The migraNon and molecular weight of the protein standards are indicated. Formula5on: 43.7 kDa Stable for one year at -20°C from date of receipt. The protein is not subject to freeze/thaw under these condiNons. (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 Size Distribu5on of Released Chroma5n: CUT&RUN was performed using CUTANA TM pAG-MNase (1:20 diluNon) with 0.5 million K-562 cells. Purified DNA was subjected to sequencing library preparaNon using an NEBNext® Ultra TM II DNA Library Prep Kit for Illumina®. Agilent Bioanalyzer traces for libraries derived from H3K4me3 CUT&RUN (blue track; ThermoFisher ScienNfic 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). Mol. Wgt. Storage and Stability: CUTANA pAG-MNase for ChIC/CUT&RUN Workflows CUTANA TM 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. Nuclease Type E. coli Expressed In Epitope Tag This product is sufficient to perform 50 CUT&RUN reacNons. Recommended use: 2.5 µL of the supplied enzyme into a 50 µL CUT&RUN reacNon (20X diluNon). For detailed applicaNons and uses of this product, please see Skene et al., 2018 (3). A detailed protocol can also be found at: EpiCypher.com/cutana-protocol 6xHis Applica5on Notes: References: This product is for in vitro research use only and is not intended for use in humans or animals. EpiCypher, Inc • Phone: 855-374-2461 • Fax: 855-420-6111 • www.epicypher.com
Transcript of CUTANA™ pAG-MNase for ChIC/CUT&RUN Workflows · 2019-08-12 · Catalog No. 15-1016 Lot No....
15-1016CatalogNo.19199003LotNo.
ProductDescrip5on:RecombinantlyproducedinE.coli,CUTANATMpAG-MNaseforChIC/CUT&RUNWorkflowsisafusionofProteinsAandGtoMicrococcalNuclease.ThisconstructisusefulinperformingChromaNnImmunocleavage(ChIC)(1)andCleavageUnderTargetsandReleaseUsingNuclease(CUT&RUN)(2,3).CUTANATMpAG-MNasecontainsaC-terminal6xHisepitopetag.
50reacNonsPackSize
150kDa 100kDa
75kDa
50kDa
37kDa
25kDa
20kDa
15kDa
ProteinGelData:CUTANATMpAG-MNase(1µg)wasresolvedviaSDS-PAGEandstainedwithCoomassieblue.ThemigraNonandmolecularweightoftheproteinstandardsareindicated.
Formula5on:
43.7kDa
Stableforoneyearat-20°Cfromdateofreceipt.Theproteinisnotsubjecttofreeze/thawunderthesecondiNons.
(1)SchmidMetal.,MolCell.2004;16:147-157(2)SkenePJ,HenikoffS.eLife2017;6:e21856(3)SkenePJetal.,NatProtoc.2018;13:1006-1019
SizeDistribu5onofReleasedChroma5n:CUT&RUNwasperformedusingCUTANATMpAG-MNase(1:20diluNon)with0.5millionK-562cells.PurifiedDNAwassubjectedtosequencinglibrarypreparaNonusinganNEBNext®UltraTMIIDNALibraryPrepKitforIllumina®.AgilentBioanalyzertracesforlibrariesderivedfromH3K4me3CUT&RUN(bluetrack;ThermoFisherScienNficCatalogNo.PA5-27029,LotSG2419844A)andH3K27me3CUT&RUN(orangetrack;CellSignalingTechnologyCatalogNo.9733,Lot14)areshown.ExcisedDNAishighlyenrichedformononucleosomes(peakat300bpreflects150bpinsertsize).
Mol.Wgt.
StorageandStability:
CUTANApAG-MNaseforChIC/CUT&RUNWorkflows
CUTANATMpAG-MNaseisprovidedasa20Xstockin10mMTrisHClpH7.5,150mMNaCl,1mMEDTA,and50%glycerol.
NucleaseType E.coliExpressedIn
EpitopeTag
Thisproductissufficienttoperform50CUT&RUNreacNons.Recommendeduse:2.5µLofthesuppliedenzymeintoa50µLCUT&RUNreacNon(20XdiluNon).FordetailedapplicaNonsandusesofthisproduct,pleaseseeSkeneetal.,2018(3).Adetailedprotocolcanalsobefoundat:EpiCypher.com/cutana-protocol
6xHis
Applica5onNotes:
References:
Thisproductisforinvitroresearchuseonlyandisnotintendedforuseinhumansoranimals.
EpiCypher,Inc•Phone:855-374-2461•Fax:855-420-6111•www.epicypher.com
CUT&RUN Data: Sequencing tracks obtained using CUTANATM pAG-MNase. (A) CUT&RUN was performed using 0.5million K-562 cells with H3K4me3 anLbody (ThermoFisher ScienLfic Catalog No. PA5-27029, Lot SG2419844A).Reference ChIP-seq tracks from ENCODE using 20 million cells (GEO Accession GSM2534288, Run SRR5339104 atlocus chr1:26,732,009-26,900,000) are shown for comparison. (B) CUT&RUN was performed using 0.5 million K-562cells with H3K27me3 anLbody (Cell Signaling Technology Catalog No. 9733, Lot 14). Reference ChIP-seq tracks fromENCODE using 20 million cells (GEO Accession GSM733658, combined Runs SRR227389 + SRR227390 at locuschr20:35,914,690-36,600,000) are shown for comparison. Total read depth for each experiment prior to alignmentis indicated on the browser tracks. In both cases, CUTANATM pAG-MNase achieved superior sensiLvity and signal-to-noisewith>10-foldreducedsequencingdepthand40-foldlesscellinput.
FormoreinformaLonregardingtheChIP-seqmethodsandENCODEresource,seeRametal.,Cell2011;147:1628-1639andNature2012;489:57-74.
Applica1on Notes Addendum: Since CUT&RUN has lower background and is compaLble with fewer cells compared to ChIP-seq, it is not recommended to assess fragment size distribuLon using agarose gel or capillary electrophoresis (e.g. AgilentBioanalyzer or TapeStaLon) prior to library preparaLon. This analysis is not indicaLve of the success of a CUT&RUNexperiment, and further the amount of DNA recovered may be below the sensiLvity of detecLon for these approaches. Togauge the success of a CUT&RUN experiment, assess DNA yield compared to + and - IgG controls, determine fragment sizedistribuLon of sequence-ready libraries, and evaluate peak structure and expected genome-wide distribuLon in NGS data. AsperSkeneetal,pg.1014step37:“targetedDNArecoveredistoolowinamountandtoosmallinsizetobedetectedbygel...”
Thisproductisforinvitroresearchuseonlyandisnotintendedforuseinhumansoranimals.
EpiCypher,Inc•Phone:855-374-2461•Fax:855-420-6111•www.epicypher.com
