CUTANA™

CUTANA™ CUT&Tag Kit

$2,695.00
SKU: 14-1102
Pack Size: 48 Reactions
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Description

The CUTANA™ CUT&Tag Kit offers a comprehensive solution for ultra-sensitive mapping of histone post-translational modifications (PTMs) [Kaya-Okur et al., 2019]. This 48 reaction kit uses an exclusive Direct-to-PCR strategy to go from cells to PCR amplified sequencing libraries in one tube, bypassing traditional library prep and minimizing sample loss [Kaya-Okur et al., 2020]. The protocol is also designed for compatibility with multi-channel pipetting for increased throughput and reproducibility. Positive (H3K27me3) and negative (IgG) control antibodies are paired with the SNAP-CUTANA™ K-MetStat Panel of nucleosome spike-in controls (Figure 2) to continuously monitor workflows and guide troubleshooting.

The recommended input for CUT&Tag is 100,000 native nuclei per reaction. Comparable data can be generated down to 10,000 nuclei, and the protocol is also validated for whole cells, cryopreserved samples, and lightly cross-linked nuclei or cells. CUT&Tag provides robust profiling for histone PTMs. For chromatin-associated proteins (e.g. transcription factors), CUTANA™ CUT&RUN is recommended (EpiCypher 14-1048, EpiCypher 14-1001/14-1002).

Validation Data

14-1102-distribution-analysis (Click to enlarge)

Figure 1: CUT&Tag DNA fragment size distribution analysis
CUT&Tag was performed as described in Figure 5. Library DNA was analyzed by Agilent TapeStation®, which confirmed that mononucleosomes were predominantly enriched in CUT&Tag (peak between 300-400 bp). Peak between 500-700 bp represents dinucleosomes.

14-1102-spike-in-controls (Click to enlarge)

Figure 2: SNAP-CUTANA™ K-MetStat Spike-in controls
DNA-barcoded designer nucleosomes (dNucs) representing 16 different K-methyl PTM states: mono-, di-, and tri-methylation at H3K4, H3K9, H3K27, H3K36, and H4K20, as well as unmodified control, were spiked into CUT&Tag reactions prior to the addition of the control antibodies provided with the kit (IgG, H3K27me3). After sequencing, instances of each spike-in barcode recovered in the CUT&Tag reactions were counted and normalized from raw fastq files using the shell script and analysis excel sheet available on the spike-in product page (EpiCypher 19-1002). Barcodes for IgG (top; normalized to the sum of total reads) and H3K27me3 (bottom; normalized to on-target) antibodies provided with this kit are shown. The spike-ins confirmed optimal experimental conditions (H3K27me3 antibody specifically recovered the target dNuc, while IgG showed no preferential enrichment).

14-1102-genome-wide-heatmaps (Click to enlarge)

Figure 3: CUT&Tag genome-wide heatmaps
CUT&Tag was performed as described in Figure 5. Heatmaps show two replicates (“Rep”) of IgG, H3K27me3, H3K4me1, and H3K4me3 antibodies in aligned rows ranked by intensity (top to bottom) relative to the H3K4me3 Rep 1 reaction. High, medium, and low intensity are shown in red, yellow, and blue, respectively. Antibodies to histone PTMs showed expected enrichment patterns and high reproducibility. H3K4me3, a marker of active transcription localized to TSSs, shows oppositional enrichment to H3K27me3 (a marker of repressive chromatin), while H3K4me1 signal flanks TSSs. IgG shows low background enrichment.

14-1102-gene-browser-tracks (Click to enlarge)

Figure 4: Representative gene browser tracks
CUT&Tag was performed as described in Figure 5. A representative 186 kb window at the LAMC3 gene is shown for two replicates (“Rep”) of IgG and H3K27me3 kit control antibodies. Representative tracks are also shown for two replicates of H3K4me1 and H3K4me3 antibodies. The CUT&Tag kit produced the expected genomic distribution for each target. Images were generated using the Integrative Genomics Viewer (IGV, Broad Institute).

cut-and-tag-methods (Click to enlarge)

Figure 5: CUT&Tag methods
CUT&Tag was performed using the CUTANA™ CUT&Tag Kit starting with 100k K562 cells and 0.5 µg of either IgG (EpiCypher 13-0042t), H3K27me3 (EpiCypher 13-0055t), H3K4me1 (EpiCypher 13-0057), or H3K4me3 (EpiCypher 13-0041*) antibodies. Libraries were run on an Illumina NextSeq2000 with paired-end sequencing (2x50 bp). Sample sequencing depth was 6.3/4.7 million reads (IgG Rep 1/Rep 2), 4.1/4.6 million reads (H3K27me3 Rep 1/Rep 2), 4.7/5.0 million reads (H3K4me1 Rep 1/Rep 2), and 5.0/4.5 million reads (H3K4me3 Rep 1/Rep 2). Data were aligned to the hg19 genome using Bowtie2. Data were filtered to remove duplicates, multi-aligned reads, and ENCODE DAC Exclusion List regions.
*EpiCypher 13-0041 does not currently meet our “SNAP-Certified in CUT&Tag” efficiency standards for robust profiling down to 10k nuclei, but is specific at 100k nuclei.

Kit Contents

Item Cat. No.
CUTANA™ pAG-Tn5 for CUT&Tag 15-1017
H3K27me3 Antibody, SNAP-Certified™ for CUT&RUN and CUT&Tag 13-0055t
CUTANA™ Rabbit IgG CUT&RUN Negative Control Antibody 13-0042t
Anti-Rabbit Secondary Antibody for CUTANA™ CUT&Tag Workflows 13-0047t
SNAP-CUTANA™ K-MetStat Panel 19-1002t
CUTANA™ Concanavalin A Conjugated Paramagnetic Beads 21-1401
CUTANA™ Non-Hot Start 2X PCR Master Mix for CUT&Tag 15-1018
CUTANA™ CUT&RUN 8-strip 0.2 mL Tubes 10-0009t
4.5 M NaCl 21-1013
0.5 M EDTA 21-1014
1 M MgCl2 21-1015
SDS Release Buffer 21-1017
SDS Quench Buffer 21-1018
0.1X TE Buffer 21-1019
Pre-Wash Buffer 21-1020
Pre-Nuclear Extraction Buffer 21-1021
Bead Activation Buffer 21-1022
5% Digitonin 21-1023
1 M Spermidine 21-1024
SPRIselect reagent manufactured by Beckman Coulter, Inc 21-1404
Multiplexing Primers 14-1102 and 14-1103 each contain combinatorial dual indices for multiplexed sequencing of up to 48 reactions. Combine the kits to multiplex up to 96 reactions.

Recommended Accessory Products

Item Cat. No.
Anti-Mouse Secondary Antibody for CUTANA™ CUT&Tag Workflows 13-0048
SNAP-CUTANA™ K-MetStat Panel 19-1002
CUT&Tag Antibodies See the list
Magnetic Separation Rack, 0.2 mL Tubes 10-0008
Magnetic Separation Rack, 1.5 mL Tubes 10-0012

Technical Information

Storage
OPEN KIT IMMEDIATELY and store components at room temperature, 4°C, and -20°C as indicated (see Kit Manual). Stable for 6 months upon date of receipt.
Instructions for Use
See Kit Manual

Additional Information

Beckman Coulter, the stylized logo, and SPRIselect are trademarks or registered trademarks of Beckman Coulter, Inc. in the United States and other countries.

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