CUTANA™ CUT&Tag Kit – 48 Reactions (Primer Set 1)
Select Multiplex Primers:
- Primer Set 1
- Primer Set 2
73 in stock
Powered by BiozThe 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 H3K4me3) 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).
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.
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 samples prior to the addition of the control antibodies provided with the kit (IgG, H3K27me3, H3K4me3). 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), H3K27me3 (middle; normalized to on-target), and H3K4me3 (bottom; normalized to on-target) antibodies provided with this kit are shown. The spike-ins confirmed optimal experimental conditions (H3K27me3 and H3K4me3 antibodies specifically recovered the target dNuc, while IgG showed no preferential enrichment).
Figure 3: CUT&Tag genome-wide heatmaps
CUT&Tag was performed as described in Figure 5. Heatmaps show two replicates (“Rep”) of IgG 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 transcription start sites (TSSs), shows enrichment consistent at TSSs, as expected. IgG shows low background enrichment.
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, H3K27me3, and H3K4me3 kit control antibodies. Representative tracks are also shown for two replicates of H3K4me1 antibody. The CUT&Tag kit produced the expected genomic distribution for each target. Images were generated using the Integrative Genomics Viewer (IGV, Broad Institute).
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), H3K4me3 (EpiCypher 13-0060t), or H3K4me1 (EpiCypher 13-0057) antibodies. Libraries were run on an Illumina NextSeq2000 with paired-end sequencing (2×50 bp). Sample sequencing depth was 5.3/4.1 million reads (IgG Rep 1/Rep 2), 11.2/9.0 million reads (H3K27me3 Rep 1/Rep 2), 8.6/5.0 million reads (H3K4me3 Rep 1/Rep 2), and 4.1/10.3 million reads (H3K4me1 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.
Figure 6: CUT&RUN methods
CUT&RUN was performed on 500k and 50k K562 cells with the SNAP-CUTANA™ K-MetStat Panel (EpiCypher 19-1002) spiked-in prior to the addition of 0.5 μg of either H3K4me3 or IgG negative control (EpiCypher 13-0042) antibodies. The experiment was performed using the CUTANA™ ChIC/CUT&RUN Kit v3 (EpiCypher 14-1048). Library preparation was performed with 5 ng of CUT&RUN enriched DNA (or the total amount recovered if less than 5 ng) using the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001/14-1002). Both kit protocols were adapted for high throughput Tecan liquid handling. Libraries were run on an Illumina NextSeq2000 with paired-end sequencing (2×50 bp). Reaction sequencing depth was 8.3 million reads (IgG 500k cell input), 15.5 million reads (IgG 50k cell input), 9.8 million reads (H3K4me3 500k cell input) and 8.6 million reads (H3K4me3 50k cell input). Data were aligned to the hg19 genome using Bowtie2. Data were filtered to remove duplicates, multi-aligned reads, and ENCODE DAC Exclusion List regions.