CUT&RUN Core Set

Cell Harvest

• Harvest cells for each sample at RT
• Wash cells 4 x with 1 mL Wash Buffer

Prepare CUT&RUN Concanavalin A Beads

• Pipette 10 µL CUT&RUN Concanavalin A Beads slurry for each sample into a tube
• Place the tubes on a magnet separator and remove the liquid carefully
• Remove the tubes from the magnetic separator
• Wash beads 3 more times with 1 mL Binding Buffer
• Finally resuspend the beads with 10 µL Binding Buffer per sample

Cell Immobilization – binding to CUT&RUN Concanavalin A Beads

• Carefully vortex the samples and add 10 µL of the prepared CUT&RUN Concanavalin A Beads to each sample
• Close tubes tightly and rotate for 5-10 min at RT

Cell Permeabilization and Primary Antibody Binding

• Place the tubes on a magnetic separator and remove the liquid carefully
• Remove the tubes from the magnetic separator
• Place each tube on the vortex mixer set to a low speed and add 100 µL Antibody Buffer containing Digitonin
• Gently vortex the tubes until the beads are resuspended
• Add 5 µL CUT&RUN anti-DYKDDDDK antibody or CUT&RUN Positive Control or CUT&RUN Negative Control corresponding to a 1:20 dilution
• Add 1 µL primary rabbit antibody against your protein of interest corresponding to a 1:100 dilution to the remaining samples
• Rotate the tubes for 2 h at RT or 4 h to O/N at 4 °C
• Place the tubes on a magnet separator and remove the liquid carefully
• Remove the tubes from the magnetic separator
• Resuspend pellet with 1 mL Digitonin Wash Buffer and mix by inversion
• Wash again

Secondary Antibody Binding (optional)
If no secondary antibody is used proceed directly to pA/G-MNase-Binding.

• Place the tubes on a magnet separator and remove the liquid carefully
• Remove the tubes from the magnetic separator
• Vortex the samples at low speed and add 100 µL Digitonin Wash Buffer per sample
• Add 5 µL Secondary Antibody corresponding to a 1:20 dilution
• Rotate the tubes for 1 h at 4 °C
• Place the tubes on a magnet separator and remove the liquid carefully
• Remove the tubes from the magnetic separator
• Resuspend with 1 mL Digitonin Wash Buffer and mix by inversion
• Wash again

Protein A-MNase or Protein A/G-MNase Binding

• Place the tubes on a magnet separator and remove the liquid carefully
• Remove the tubes from the magnetic separator
• Place each tube on the vortex mixer set to a low speed and add 50 µL Digitonin Wash Buffer and 2.5 µL pA/G-MNase per sample
• Rotate the tubes for 1 h at 4 °C
• Place the tubes on a magnet separator and remove the liquid carefully
• Remove the tubes from the magnetic separator
• Resuspend with 1 mL Digitonin Wash Buffer and mix by inversion
• Wash again

MNase Digestion and Release of pA/G-MNase-Bound Chromatin Fragments

High Ca2+/Low Salt Chromatin Cleavage

• Quick pulse in a table-top centrifuge (max 100 x g)
• Place the tubes on a magnet separator and remove the liquid carefully
• Resuspend with 1 mL Low-Salt Rinse Buffer and mix by inversion
• Quick pulse in a table-top centrifuge (max 100 x g)
• Place the tubes on a magnet separator and remove the liquid carefully
• Wash again
• Place each tube on the vortex mixer set to a low speed and add 200 µL ice cold Low Salt Incubation Buffer per sample
• Incubate tubes at 0 °C for 5 min
• Place the tubes on a cold magnet separator and remove the liquid carefully
• Remove the tubes from the magnetic separator
• Resuspend with 200 µL Low Salt Stop Buffer and mix by gentle vortexing
• Incubate tubes at 37 °C for 30 min
• Place the tubes on a magnet separator
• Transfer the supernatant containing the pA/G-MNase-bound digested chromatin fragments to fresh 1.5 mL tubes
• Proceed with DNA extraction