CUT&RUN Pro Set

• Cell harvest
• • Harvest 300,000 murine endothelial cells for each sample at RT. Keep cells for each sample in separate tubes.
  • Centrifuge cell solution 3 min at 600 x g at RT.
  • Remove the liquid carefully.
  • Gently resuspend cells in 1 ml Wash Buffer by pipetting and transfer cell solution to a 1.5 ml microcentrifuge tube.
  • Centrifuge cell solution 3 min at 600 x g at RT and discard the supernatant.
  • Repeat three times for a total of four washes.
  • Resuspend cell pellet for each sample in 1 ml Wash Buffer by gently pipetting.
• Concanavalin A beads preparation
• • Prepare one 1.5 ml microcentrifuge tube for each sample.
  • Gently resuspend the CUT&RUN Concanavalin A Beads.
  • Pipette 10 µl CUT&RUN Concanavalin A Beads slurry for each sample into the 1.5 ml microcentrifuge tubes.
  • Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
  • Remove the microcentrifuge tube from the magnetic stand.
  • Pipette 1 ml Binding Buffer into each tube and resuspend CUT&RUN Concanavalin A Beads by gentle pipetting.
  • Spin down the liquid from the lid with a quick pulse in a bench-top centrifuge.
  • Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
  • Remove the microcentrifuge tube from the magnetic stand.
  • Repeat twice for a total of three washes.
  • Gently resuspend the CUT&RUN Concanavalin A Beads in a volume of Binding Buffer corresponding to the original volume of bead slurry, i.e. 10 µl per sample.
• Cell immobilization – binding to Concanavalin A beads
• • Carefully vortex the cell suspension and add 10 µl of the CUT&RUN Concanavalin A Beads in Binding Buffer to each sample.
  • Close tubes tightly and rotate for 10 min at RT.
• Cell permeabilization and primary antibody binding
• • Place the microcentrifuge tubes on a magnetic stand until the fluid is clear. Remove the liquid carefully.
  • Remove the microcentrifuge tubes from the magnetic stand.
  • Place each tube at a low angle on the vortex mixer set to a low speed and add 100 µl Antibody Buffer containing digitonin.
  • Gently vortex the microcentrifuge tubes until the beads are resuspended.
  • Add 2 µl H3K27me3 positive control antibody ABIN6923144 corresponding to a 1:50 dilution.
  • Rotate the microcentrifuge tubes for O/N at 4 °C.
  • Spin down the liquid and place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
  • Remove the microcentrifuge tubes from the magnetic stand.
  • Resuspend with 1 ml Digitonin Wash Buffer and mix by inversion. If clumps occur, gently remove the clumps with a 1 ml pipette tip.
  • Repeat once for a total of two washes.
• pAG-MNase Binding
• • Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
  • Remove the microcentrifuge tubes from the magnetic stand.
  • Vortex the sample at low speed and add 50 μl Digitonin Wash Buffer per sample along the side of the tube. Add 2.5µl CUTANA™ pAG-MNase for ChIC/CUT&RUN Assays (ABIN6950951, lot 19199003).
  • Rotate the microcentrifuge tubes for 1 h at 4 °C.
  • Spin down the liquid and place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
  • Remove the microcentrifuge tubes from the magnetic stand.
  • Resuspend with 1 ml Digitonin Wash Buffer and mix by inversion. If clumps occur, gently remove the clumps with a 1 ml pipette tip.
  • Repeat once for a total of two washes.
• MNase digestion and release of pAG-MNase-antibody-chromatin complexes
• • Spin down the liquid from the lid with a quick pulse in a table-top centrifuge.
  • Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
  • Resuspend with 1 ml Low Salt Rinse Buffer and mix by inversion. If clumping occurs, gently remove the clumps with a 1 ml pipette tip.
  • Spin down the liquid from the lid with a quick pulse in a table-top centrifuge.
  • Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
  • Repeat once for a total of two washes.
  • Place each tube at a low angle on the vortex mixer set to a low speed and add 200 μl ice cold Low Salt Incubation Buffer per sample along the side of the tube.
  • Incubate tubes at 0 °C for 1 h.
  • Place the tubes on a cold magnet stand until the fluid is clear. Remove the liquid carefully.
  • Remove the microcentrifuge tubes from the magnetic stand.
  • Resuspend with 200 µl Low Salt Stop Solution and mix by gentle vortexing.
  • Incubate tubes at 37 °C for 30 min.
  • Place the tubes on a magnet stand until the fluid is clear.
  • Transfer the supernatant containing the pAG-MNase-bound digested chromatin fragments to fresh 1.5 ml microcentrifuge tubes.
• DNA extraction
• • Add 2 µl 10% SDS to a final concentration of 0.1% and 5 µl Proteinase K (10 mg/ml) to a final concentration of 0.25 mg/ml to each supernatant containing the pAG-MNase-bound digested chromatin fragments.
  • Gently vortex tubes at a low speed of approximately 1100 rpm.
  • Incubate tubes at 37 °C O/N.
  • Add 200 µl Phenol-Chloroform-Isoamyl alcohol (PCI) to tube.
  • Vortex tubes thoroughly at high speed until the liquid appears milky.
  • Transfer liquid to a phase-lock tube.
  • Centrifuge tubes in a tabletop centrifuge at 16000 x g at RT for 5 min.
  • Carefully transfer the upper aqueous phase to a fresh 1.5 ml microcentrifuge tube containing 200 µl chloroform:isoamyl alcohol 24:1 solution.
  • Vortex tubes thoroughly at high speed until the liquid appears milky.
  • Centrifuge tubes in a benchtop centrifuge at 16000 x g at 4 °C for 5 min.
  • Carefully transfer to upper aqueous phase to a fresh 1.5 ml microcentrifuge tube containing 2 µl glycogen (diluted 1:10 to 2 mg/ml from the 20 mg/ml stock solution).
  • Add 20 µl 3 M NaOAc pH 5.2 or 150 µl 5 M NH₄OAc.
  • Add 500 µl 100% ethanol.
  • Place O/N at -20 °C.
  • Centrifuge tubes in a benchtop centrifuge at 16000 x g at 4 °C for 5min.
  • Remove the liquid carefully with a pipette.
  • Add 1ml 70% ethanol.
  • Centrifuge tubes in a benchtop centrifuge at 16000 x g at 4 °C for 1 min.
  • Remove the liquid carefully with a pipette.
  • Air-dry the pellet, then dissolve in 15 µl 1 mM Tris-HCl, 0.1 mM EDTA.
• Sequencing library preparation
• • Sequencing libraries were prepared using the KAPA HyperPrep Kit (Kapa Biosystems, KR0961) according to the manufacturer’s recommendations with the following modification:
  • For the post-ligation cleanup kit, the SPRI bead to ligation reaction ratio was increased to 1.1 to avoid loss of CUT&RUN products.
  • The PCR conditions were optimized for short products to avoid melting of the small fragments during elongation and favor short PCR products:
  • Initial denaturation: 1 cycle: 45 sec at 98 °C
  • Amplification: 16 cycles: 15 sec at 98 °C, followed by 10 sec at 60 °C
  • Final extension: 1 cycle for 1 min at 72°C
• Sample quality control
• • Evaluate DNA fragmentation via Bionanalyzer Electrophoresis before and after library preparation.

MNase digestion was tested for 5 min, 15 min, 45 min, and 1 h. DNA from the 1 h digestion reaction was selected for library preparation because of the higher ratio of mononucleosomal fragments.