Depending on your target, you may need to consider modifications to the CUTANA™ CUT&RUN DNA Purification protocol.
The current CUTANA™ CUT&RUN DNA Purification protocol uses a 1.4X SPRI bead ratio to capture mononucleosome-sized chromatin fragments (~140-180 bp). In EpiCypher’s experience, the majority of transcription factors, modifying enzymes, and other chromatin-associated proteins generate predominantly mononucleosome-sized fragments in CUT&RUN, and are successfully profiled using the current protocol.
However, a few select targets, such as some transcription factors and nuclear hormone receptors, generate both mononucleosome- and subnucleosome-sized fragments (<120 bp). To avoid a loss in yields, these targets may benefit from a modified CUT&RUN DNA purification strategy, using either 1.8X SPRI or spin columns.
These modifications are outlined below. If you are unsure what to expect for your target, reach out to EpiCypher Tech Support for guidance.
Option 1: Increase the ratio of SPRI beads during purification.
Use a 1.8X volume of SPRI reagent for purification of CUT&RUN-enriched DNA, which will enhance capture of smaller fragments. Perform Ethanol washes and elution as described in the CUT&RUN protocol.
Note: This will require purchase of additional SPRI beads, as our Kit is optimized for 1.4X SPRI ratios. Additional beads are available via purchase of our CUTANA™ Quick Cleanup DNA Purification Kit (EpiCypher 14-0052).
Option 2: Use spin columns for DNA purification.
Purify CUT&RUN-enriched DNA using spin columns (such as NEB Monarch® Spin PCR & DNA Cleanup Kit, T1130) rather than SPRI beads. If you choose this method, follow the protocol provided with the spin column kit!
Bonus: Additional Transcription Factor CUT&RUN Controls
If you routinely profile transcription factors in CUT&RUN, you may consider using our validated CTCF antibody (EpiCypher 13-2014) as a positive control, in addition to the H3K4me3 and H3K27me3 positive controls provided with the CUTANA CUT&RUN Kit.