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14-1102-48s1

CUTANA^™ CUT&Tag Kit with Primer Set 1

48 Reactions

14-1102

14-1102-48s2

CUTANA^™ CUT&Tag Kit with Primer Set 2

48 Reactions

14-1103

14-1102-24s3

CUTANA^™ CUT&Tag Kit with Primer Set 1 Subset

24 Reactions

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 0.5µg 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 (2x50 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.