CUTANA™ pAG-Tn5 for CUT&Tag
Powered by BiozCUTANA pAG-Tn5 is the key reagent for performing efficient mapping of chromatin features in Cleavage Under Targets and Tagmentation (CUT&Tag) [Kaya-Okur et al., 2019]. CUT&Tag offers significant improvements in signal to noise at reduced cell inputs and sequencing depth compared to ChIP-seq. As a fusion of Proteins A and G to a highly active E. coli transposase mutant (Tn5), CUTANA pAG-Tn5 is compatible with target antibodies from a broad spectrum of host species. It also comes without an epitope tag, making it suitable for tag-mediated CUT&Tag (e.g. FLAG, HA, TY1, V5, etc.). This product is highly purified to remove contaminating E. coli DNA, which can be tagmented during storage and complicate analysis at low cell numbers. For superior normalization as well as antibody validation and reaction monitoring, SNAP-CUTANA™ nucleosome spike-ins are recommended. The Tn5 comes charged with mosaic adapters and is ready to be used in CUT&Tag.
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
Figure 1: Protein gel data
CUTANA pAG-Tn5 for CUT&Tag (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 191 kDa.
Figure 2: Size distribution of released chromatin
CUT&Tag was performed as described above. Recovered DNA was directly PCR amplified to produce sequence-ready libraries. Agilent TapeStation traces for libraries derived from negative control Rabbit IgG (top) 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 pAG-Tn5 show a 232 kb close up view of the LAMC3 gene. CUTANA pAG-Tn5 produced clear peaks with genomic distribution profiles consistent with the known biological functions of H3K4me3 and H3K27me3 as well as minimal background in the IgG negative control.
CUT&Tag methods
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™ pAG-Tn5 (1:20 dilution) following the EpiCypher Direct-to-PCR CUT&Tag protocol. Libraries were run on an Illumina NextSeq2000 with paired-end sequencing (2×50 bp). Sample sequencing depth was 0.3 million reads (IgG), 1.7 million reads (H3K4me3), and 3.8 million reads (H3K27me3). Data were aligned to the hg19 genome using Bowtie2. Data were filtered to remove duplicates, multi-aligned reads, and ENCODE DAC Exclusion List regions.
- Type: Transposase
- Mol Wgt: 191 kDa
- Host: E. coli
- Epitope Tag: None
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’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3’
Tn5ME-B: 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3’
Tn5ME-rev: 5’-[phos]CTGTCTCTTATACACATCT-3’
Application Notes
Add 2.5 µL of the supplied enzyme to a 50 µL CUT&Tag reaction (20X dilution). For detailed applications and uses of this product, please see our CUT&Tag protocol.
| Item | Cat. No. |
|---|---|
| CUTANA™ Concanavalin A Conjugated Paramagnetic Beads | 21-1401 |
| SNAP-CUTANA™ K-MetStat Panel | 19-1002 |
| H3K4me3 Antibody, SNAP-Certified™ for CUT&RUN and CUT&Tag | 13-0060 |
| CUTANA™ Rabbit IgG CUT&RUN Negative Control Antibody | 13-0042 |
| 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 |
| 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 |
| CUTANA™ Nuclei Extraction Buffer | 21-1026 |
| CUTANA™ Protease Inhibitor Tablets | 21-1027 |
