Digestion of CUT&RUN Input DNA by MNase

Fragmentation of input DNA is required for compatibility with downstream NG-Sequencing but is not necessary for downstream qPCR analysis. If a sonicator is not available, we recommend using the un-fragmented input DNA for qPCR analysis; however, the input DNA should be purified using phenol/chloroform extraction and ethanol precipitation because the size of un-fragmented input DNA is too large to be purified using DNA spin columns. If a sonicator is not available and downstream NG-Sequencing analysis is desired, one can use the CUT&RUN normal IgG antibody sample as the negative control, although this is not ideal because the normal IgG-enriched sample may show non-specific DNA enrichment. Alternatively, an input DNA fragmentation protocol using MNase is available as below.

NOTE: The following reagents are required for CUT&RUN input DNA digestion and are not included in our CUT&RUN Assay Kit #86652 or our CUT&RUN Assay Kit (with Drosophila Spike-In Control) #84647: SimpleChIP^® Enzymatic Cell Lysis Buffers A & B #14282, Micrococcal Nuclease #10011, DTT (Dithiothreitol) #7016, 0.5 M EDTA #7011, 10% SDS Solution #20533, DNA Purification Buffers and Spin Columns (ChIP, CUT&RUN) #14209, and Nuclease-free Water #12931. If one is not using our CUT&RUN Assay Kit #86652 or our CUT&RUN Assay Kit (with Drosophila Spike-In Control) #84647, please also purchase the following reagents separately: Concanavalin A Magnetic Beads and Activation Buffer #93569, Protease Inhibitor Cocktail (200X) #7012, Proteinase K (20 mg/mL) #10012, and RNAse A (10 mg/mL) #7013.

Before Starting:

! All buffer volumes should be increased proportionally based on the number of input DNA samples.

NOTE: For all incubation steps, it is not necessary to mix samples by rocking or rotation. Simply place the tubes in a rack at the designated temperatures. Mixing does not improve assay performance and may instead cause bead clumping or bead loss due to sticking on tube walls and caps.

• Place Concanavalin A Bead Activation Buffer on ice.
• Remove and warm 200X Protease Inhibitor Cocktail (PIC) #7012. Make sure it is completely thawed prior to use.
• Prepare 1 M DTT (192.8 mg DTT #7016 + 1.12 mL dH₂O). Make sure DTT crystals are completely in solution.

  !! IMPORTANT: Once in solution, store 1M DTT at -20°C.

• Prepare 1 mL of 1X Buffer A (250 µL 4X Buffer A #7006 + 750 µL Nuclease-free Water #12931 + 0.5 µL 1M DTT + 5 µL 200X PIC) per input sample and place it on ice.
• Prepare 1.2 mL of 1X Buffer B (300 µL 4X Buffer B #7007 + 900 µL Nuclease-free Water #12931 + 0.6 µL 1M DTT) per input and place it on ice.

1. Carefully resuspend Concanavalin A Magnetic Beads by gently pipetting up and down, making sure not to splash any bead suspension out of the tube. Transfer 10 µL of the bead suspension per input sample to a new 1.5 mL microcentrifuge tube.

   NOTE: Avoid vortexing of the Concanavalin A Magnetic Bead suspension as repeated vortexing may displace the Concanavalin A from the beads.

2. Add 100 µL Concanavalin A Bead Activation Buffer per 10 µL beads. Gently mix beads by pipetting up and down.
3. Place the tube on a magnetic rack until the solution becomes clear (30 sec to 2 min) and then remove the liquid.

   NOTE: To avoid loss of beads, remove liquid using a pipet. Do not aspirate using a vacuum.

4. Remove tubes from the magnetic rack. Wash the beads a second time by repeating steps 2 and 3.
5. Add a volume of Concanavalin A Bead Activation Buffer equal to the initial volume of bead suspension added (10 µL per sample) and resuspend by pipetting up and down.
6. Add 10 µL of activated bead suspension to each input sample prepared in Section II of the CUT&RUN Assay Kit #86652 protocol or the CUT&RUN Assay Kit (with Drosophila Spike-In Control) #84647 protocol.
7. Incubate the sample for 5 min at room temperature.
8. Briefly centrifuge the sample at 100 x g for 2 sec to remove cell:bead suspension from the cap of the tube. Place the tube on the magnetic rack until the solution turns clear (30 sec to 2 min), then remove and discard the liquid.
9. Remove the tube from the stand. Resuspend cell:bead suspension in 1 mL ice-cold 1X Buffer A + DTT + PIC per input sample. Incubate on ice for 10 min.
10. Briefly centrifuge the sample at 100 x g for 2 sec to remove cell:bead suspension from the cap of the tube. Place the tube on the magnetic rack until the solution turns clear (30 sec to 2 min), then remove and discard the liquid.
11. Resuspend cell:bead suspension in 1 mL ice-cold 1X Buffer B + DTT per input sample. Repeat Step 10 and resuspend the pellet in 100 µL 1X Buffer B +DTT per input sample.
12. Dilute 1 µL Micrococcal Nuclease #10011 1:40 by adding 39 µL of 1X Buffer B + DTT. Add 0.5 µL of diluted Micrococcal Nuclease to each input sample, mix by pipetting up and down. Incubate for 20 min at 37°C with frequent mixing to digest DNA to a length of approximately 150 bp.

    NOTE: Do NOT save the remaining diluted Micrococcal Nuclease for future experiments. Micrococcal Nuclease is not stable for long periods of time in Buffer B + DTT.

13. Stop digestion by adding 10 µL of 0.5 M EDTA #7011 per input sample.
14. Add 1 µL of 10% SDS Solution #20533 (0.1% final concentration), 2 µL of proteinase K (20 mg/mL) #10012, and 0.5 µL of RNAse A #7013 to each sample. Vortex to mix.
15. Incubate sample at 65°C for 2 hr.
16. After incubation, spin samples at 16,000g for 2 min.
17. Place the tube on the magnetic rack until the solution turns clear (30 sec to 2 min).
18. Collect supernatant and continue with Section VII-A of the CUT&RUN Assay Kit #86652 protocol or Section VIII-A of the CUT&RUN Assay Kit (with Drosophila Spike-In Control) #84647 protocol (DNA Purification Using Spin Columns) to purify input DNA.

    NOTE: During DNA spin column purification, be sure to add 550 µL of DNA Binding Buffer to each input sample to have 5 volumes of DNA Binding Buffer for every 1 volume of sample.