What are SNAP-CUTANA Spike-ins? How do they work?
What are SNAP-CUTANA™ Spike-ins?
SNAP-CUTANA Spike-in Controls are a defined spike-in control added to cell samples at the start of CUT&RUN experiments. They are specifically designed for reactions targeting histone post translational modifications (PTMs), such as histone lysine methylation, and allow users to examine histone PTM antibody performance, workflow success, and much more. Because SNAP-CUTANA Spike-ins replicate chromatin structure (i.e. nucleosomes), the natural target of CUT&RUN, they provide accurate on- and off-target substrates for histone PTM antibodies.
Features and advantages include:
- Fast: SNAP-CUTANA Spike-ins are coupled to magnetic beads, allowing them to be added to CUT&RUN reactions in one quick step for seamless workflow integration (Figure 1).
- Easy: No protocol modifications are necessary. SNAP-CUTANA Spike-in data are quickly analyzed in final sequencing data using our step-by-step instructions.
- Reliable: Use these controls to examine sample quality, MNase activity, antibody specificity, and troubleshoot challenging workflows.
Figure 1. SNAP-CUTANA Spike-in Controls are pools of highly pure nucleosomes carrying defined histone PTMs. For instance, the SNAP-CUTANA K-MetStat Panel shown in this figure is a pool of 16 nucleosomes representing 16 distinct methyl-lysine states. Spike-in nucleosomes are individually coupled to magnetic beads and pooled into a single panel for convenient one-step addition to CUT&RUN workflows. pAG-MNase cleavage releases all antibody-bound targets into solution, including antibody-bound spike-ins. Each spike-in nucleosome contains a PTM-specific DNA barcode sequence, which enables detection of K-MetStat Panel controls in sequencing data.
How do SNAP-CUTANA Spike-ins work in CUT&RUN?
You can add SNAP-CUTANA Spike-in Controls to reactions that are mapping a histone PTM included in the Panel. SNAP-CUTANA Spike-ins are added to reactions just prior to addition of target-specific antibody, in Section IV of the CUT&RUN Protocol (Figure 2).
Immobilization of spike-in nucleosomes on magnetic beads makes them similar to bead-coupled cells, allowing both to be captured using magnetic separation racks. This means that spike-ins can be added early in the experiment for side-by-side processing with bead-coupled cells, all in the same reaction tube. Spike-ins can thus report on multiple aspects of the CUT&RUN workflow, including antibody specificity and pAG-MNase activity.
Figure 2. Schematic showing how SNAP-CUTANA Spike-ins, such as the K-MetStat Panel, are used during CUT&RUN workflows.
No other modifications to the protocol are required. You simply continue the CUT&RUN workflow, adding your target specific antibody for overnight incubation. Antibodies bind their specific histone PTM target in permeabilized cells AND in the SNAP-CUTANA Spike-in Panel. The next day, pAG-MNase is used to cleave antibody-bound chromatin (in cells) and antibody-bound spike-ins.
Cleaved spike-ins are released from magnetic beads into solution along with the clipped DNA fragments from cells. A magnet is used to removed unclipped spike-ins and cells, and target fragments are purified from the supernatant for sequencing.
The PTM-specific DNA barcode sequences on the spike-in nucleosomes (Figures 1 and 2) are used to detect reads from the spike-ins vs. cells in sequencing data. Generally, we aim for ~1% of total sequencing reads to be from SNAP-CUTANA Spike-ins, although this may vary based on sequencing depth and target abundance. The main goal is to have many thousands of sequencing reads aligned to SNAP-CUTANA Spike-ins, which enables adequate sampling of the panel and reliable use in downstream applications.
When assessing the reads assigned to spike-ins, you should see enrichment for on-target PTM and minimal reads from off-target PTMs in the Panel (Figure 2). These data can be used as a direct readout for PTM recovery and assay success, described in more detail here.