CUTANA™

CUTANA™ Fluorescent pAG-Tn5 for CUT&Tag

$745.00
SKU: 15-1026
Pack Size: 125 µL
  • Type:  Transposase
  • Mol Wgt:  192 kDa
  • Host:  E. coli
  • Epitope Tag:  None

Description

Products in EpiCypher’s IDEA Toolbox (Innovation and Discovery of Epigenetic Applications) offer access to reagents without known or fully defined uses, enabling researchers to explore cutting-edge applications. Due to their novelty and unexplored potential, EpiCypher will engage in limited technical support.

CUTANA Fluorescent pAG-Tn5 is a fusion of Proteins A and G to E. coli transposase (Tn5), the key enzyme for CUT&Tag [1]. The Mosaic A and Mosaic B adapters loaded in this enzyme bear a 5’ Cy5 fluorescent dye to enable multiomic visualization and mapping of chromatin features. This product is highly purified to remove contaminating E. coli DNA. For an enzyme charged with non-fluorescent adapters that can be used in standard CUT&Tag, see EpiCypher 15-1017.

Validation Data

CUT&Tag was performed on 100k K562 cells with 0.5 µg of either IgG (EpiCypher 13-0042), H3K4me3 (EpiCypher 13-0041), or H3K27me3 (ThermoFisher MA5-11198) antibodies using CUTANA™ Fluorescent pAG-Tn5 (50 nM final; see Technical Datasheet for lot-specific recommendation) following the EpiCypher Direct-to-PCR CUT&Tag protocol. Libraries were run on an Illumina NextSeq2000 with paired-end sequencing (2x50 bp). Sample sequencing depth was 0.5 million reads (IgG), 2.5 million reads (H3K4me3), and 6.3 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: DNA charging data
CUTANA Fluorescent pAG-Tn5 for CUT&Tag (0.5 µg) was resolved via Native PAGE alongside uncharged pAG-Tn5 (EpiCypher 15-1025) and charged pAG-Tn5 (EpiCypher 15-1017). The gel was imaged and Cy5 emission was observed at 670 nm (right), stained with ethidium bromide, and imaged again at 605 nm. The observed shift indicates successful loading of the Tn5, and fluorescent imaging confirms the presence of the dye.

Figure 2: Size distribution of released chromatin
CUT&Tag was performed as described above. Recovered DNA was directly PCR amplified to produce sequenceready libraries. Agilent TapeStation traces for libraries derived from negative control Rabbit IgG (top), H3K4me3 (middle), and H3K27me3 (bottom) are shown. Excised DNA is highly enriched for mononucleosomes (peak at ~300 bp reflects ~150 bp insert size).

Figure 3: CUT&Tag data
CUT&Tag was performed as described above. Representative sequencing tracks obtained using CUTANA Fluorescent pAG-Tn5 for CUT&Tag show a 337 kb view of the TRMT2A gene. CUTANA pAG-Tn5 produced clear peaks with genomic distribution consistent with the known biological functions of H3K4me3 and H3K27me3 as well as minimal background in the IgG negative control.

Figure 4: Protein gel data
CUTANA pAG-Tn5 (1 µg) was resolved via SDS-PAGE and stained with Coomassie blue. The migration and molecular weight of the protein standards are indicated. Uncharged pAG-Tn5 monomer is 78.5 kDa, however once charged with DNA, Tn5 dimerizes to a final complex weight of 192 kDa.

Recommended Accessory Reagents

Item CAT
Anti-Rabbit Secondary Antibody for CUTANA™ CUT&Tag 13-0047
Anti-Mouse Secondary Antibody for CUTANA™ CUT&Tag 13-0048
CUTANA™ Non-Hot Start 2X PCR Master Mix for CUT&Tag 15-1018
Histone H3K4me3 Antibody, SNAP-Certified™ for CUT&RUN 13-0041
CUTANA™ Rabbit IgG CUT&RUN Negative Control Antibody 13-0042

Technical Information

Storage
Stable for one year at -20°C from date of receipt. The protein is not subject to freeze/thaw under these conditions.
Formulation
50 mM HEPES-KOH pH 7.2, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1% Triton X-100, 50% glycerol
Adapters

Tn5ME-A: 5’-[Cy5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3’

Tn5ME-B: 5’-[Cy5]GATTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3’

Tn5ME-rev: 5’-[phos]CTGTCTCTTATACACATCT-3’

Application Notes

A 20X dilution of this reagent is sufficient for a standard CUT&Tag reaction following the EpiCypher CUTANA™ Direct-to-PCR CUT&Tag protocol, indicating that addition of the fluorescent dye does not prevent Tn5-mediated tagmentation of chromatin (see Figures 2 & 3). However, application-specific optimization will be necessary for use in microscopy. Due to the novelty of this reagent, EpiCypher will not engage in application tech support.

References

Background References:
[1] Kaya-Okur et al. Nat. Commun. (2019). PMID: 31036827

Documents & Resources

Intellectual Property

US Pat. No. 10,689,643, 11,306,307, EU Pat. No. 3,688,157, 2,999,784 and related patents and pending applications

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