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CUT&RUN Pro Set Accessory Reagents

Independent Validation (1)
Catalog No. ABIN6923138
$254.00
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  • Reactivity
    Eukaryotes
    Application
    Cleavage Under Targets and Release Using Nuclease (CUT&RUN), Cleavage Under Targets and Tagmentation (CUT&Tag)
    Purpose
    This set contains Magnetic ConA Beads (Agarose) for CUT&RUN/CUT&Tag Assays, CUT&RUN Positive and Negative Control for the CUT&RUN method for improved genome-wide detection of Protein-DNA-Interactions.
    Characteristics
    CUT&RUN (Cleavage Under Targets And Release Using Nuclease) offers a new approach to pursue epigenetics.
    CUT&RUN overcomes various downfalls of ChIP-Seq with improved workflow.
    CUT&RUN-Sequencing has the advantage of being a simpler technique with lower costs due to the high signal-to-noise ratio, requiring less depth in sequencing.
    CUT&RUN has the potential to replace all ChIP-based applications.
    Components
    • CUT&RUN Positive Control (Recombinant Rabbit anti-H3K27me3 Antibody)
    • CUT&RUN Negative Control (Polyclonal Guinea Pig anti-Rabbit IgG Antibody, Pre-Adsorbed)
    • Magnetic ConA Beads (Agarose) for CUT&RUN/CUT&Tag Assays
    Material not included
  • Reagent Preparation
    • Wash Buffer
    • Binding Buffer
    • Antibody Buffer
    • Digitonin Wash Buffer
    • 2x Stop Buffer
    • Low Salt Rinse Buffer
    • Low Salt Incubation Buffer
    • Low Salt Stop Buffer
    Assay Procedure
    1. Cell Harvest

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

    2. Prepare Magnetic ConA Beads (Agarose) for CUT&RUN/CUT&Tag Assays

      • Pipette 10 µL Magnetic ConA Beads (Agarose) for CUT&RUN/CUT&Tag Assays 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

    3. Cell Immobilization – binding to Magnetic ConA Beads (Agarose) for CUT&RUN/CUT&Tag Assays

      • Carefully vortex the samples and add 10 µL of the prepared Magnetic ConA Beads (Agarose) for CUT&RUN/CUT&Tag Assays to each sample
      • Close tubes tightly and rotate for 5-10 min at RT

    4. 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 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

    5. 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

    6. 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

    7. 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
    Restrictions
    For Research Use only
  • Validation #104253 (Cleavage Under Targets and Release Using Nuclease)
    'Independent Validation' Badge
    by
    New strategies to inhibit tumor angiogenesis laboratory headed by Prof. Elisabetta Dejana, IFOM - the FIRC institute of Molecular Oncology
    No.
    #104253
    Date
    02/18/2021
    Antigen
    H3K27me3
    Lot Number
    CR0109190001
    Method validated
    Cleavage Under Targets and Release Using Nuclease
    Positive Control
    Recombinant rabbit anti-H3K27me3 antibody
    Negative Control
    Polyclonal guinea pig anti-rabbit IgG antibody
    Notes

    Passed. ABIN6923144 is suitable for CUT&RUN to prepare H3K27me3 targeted DNA fragments from genomic murine DNA.

    'Independent Validation' Badge
    Validation Images
    Full Methods
    Primary Antibody
    Secondary Antibody
    Full Protocol
    • 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.
    Experimental Notes

    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.

  • Buffer
    CUT&RUN Positive Control: 50 % Glycerol/PBS, 1 % BSA, 0.09 % (w/v) Sodium Azide
    CUT&RUN Negative Control: 0.02 M Potassium Phosphate, 0.15 M NaCl, pH 7.2, 0.01 % (w/v) Sodium Azide
    Magnetic ConA Beads (Agarose) for CUT&RUN/CUT&Tag Assays: 20 mM Sodium Acetate pH 6.6, 20 % Ethanol
    Preservative
    Sodium azide
    Precaution of Use
    This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
    Storage
    4 °C/-20 °C
    Storage Comment
    Magnetic ConA Beads (Agarose) for CUT&RUN/CUT&Tag Assays ABIN6952467 must not be frozen
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