Description

Description: The SNAP-CUTANA™ K-MetStat Panel of spike-in controls for CUT&RUN and CUT&Tag offers an all-in-one solution to determine antibody specificity for histone post-translational modifications (PTMs), monitor assay success, and normalize data for quantitative chromatin mapping. The panel contains designer nucleosomes (dNucs) representing 15 different K-methyl PTM states: mono-, di-, and trimethylation at H3K4, H3K9, H3K27, H3K36, & H4K20, as well as unmodified control (Figure 1). Each PTM is represented by two unique DNA-barcoded templates (A and B, for an internal technical replicate). Each dNuc is individually conjugated to paramagnetic beads and pooled into a single panel for convenient one-step spike-in to CUT&RUN and CUT&Tag experiments. The panel is added to ConA-immobilized cells prior to the addition of an antibody targeting a histone lysine methylation state or IgG negative control (see Application Notes and Table 1). pAG-MNase-mediated release or pAG-Tn5-mediated tagmentation of genomic chromatin and the barcoded nucleosomes is dependent on the specificity of the antibody used. After sequencing, the relative read count of each spike-in nucleosome barcode provides a quantitative metric of on- vs. off-target recovery (Figures 4 and 5) as well as quantitative sample normalization, thereby gauging experimental success, guiding troubleshooting efforts, and enabling reliable cross-sample comparisons.

Additional Text

Application Notes

Product Use: Use the SNAP-CUTANA™ K-MetStat Panel for control reactions containing positive (e.g. H3K4me3) and negative (IgG) antibodies, as well as samples with an antibody to any of the 15 lysine methyl states in the K-MetStat Panel. Just before antibody addition, pipette to resuspend beads (do not vortex), then spike in 2 µL per 500k cells for CUT&RUN and 2 µL per 100k cells for CUT&Tag. If using less than the standard number of cells, decrease the amount of SNAP-CUTANA spike-in linearly by preparing a "working stock" dilution of the panel in the appropriate buffer, made fresh the day of use (Table 1). Adjust spike-in volume as needed aiming for the spike-in barcodes to comprise ~1% of the total unique sequencing reads. Expect higher for low abundance targets / negative controls (H3K4me3; ~1-10% / IgG; ~10-20%) and lower for high abundance targets (H3K27me3; 0.1-1%). Table 1 gives recommended dilution amounts for varying numbers of starting cells, but optimization may be required for user-specific conditions.

Storage Note

Store at -20°C. Lower temperatures can cause freezing and will permanently damage the magnetic beads. Stable for six months from date of receipt. Pipette to resuspend beads; DO NOT VORTEX.