TITLE: CUT &amp; RUN
AUTHORS: Yunkyeong Lee

[DESCRIPTION]
CUT & RUN protocol

[STEPS]
1. EpiCypher CUTANATM ChIC/CUT&RUN kit (Kit Version 2.0, User Manual Version 2.1)
Catalog No. 14-1048, 48 ChIC/CUT&RUN samples


Overview of the CUTANA CUT&RUN protocol


1. Immobilize & permeabilize cells (or nuclei)
2. Add antibody to histone PTM or chromatin-interacting protein
3. Add & activate pAG-MNase to cleave target-DNA complex
4. Target-DNA complex diffuses out, collect supernatant
5. Extract DNA & prepare sequencing library
6. Next-generation sequencing and data analysis




BOX 1 (at RT under the PCR machine): DNA Binding Buffer (25 mL), DNA Wash Buffer (5 mL), EDTA 100 mM (500 uL), CaCl2(500 uL), DNA Elution Buffer (1.2 mL), DNA Cleanup Columns (50 Columns), DNA Collection Tubes (50 Tubes), 8-Strip Tubes (56 Tubes) 

BOX 2 (at 4°C under the PCR machine): SA Beads (40 uL) (X), ConA Beads (550 uL) -> (Jan 2023 Re-ordering), Stop Buffer (1.5 mL), Bead Activation Buffer (12 mL), Pre-Wash Buffer (105 mL)

BOX 3 (at-20 °C in Franklin(Nicole's)): IgG Negative Control (10 uL), H3K4me3 Positive Control (10 uL), Spermidine (100 uL), pAG-MNase (48 runs), Spike-in DNA (100 ng), 5% Digitonin (100 uL)


+ EpiCypher 50 uL - 0.2 mL Magnetic Rack, Invivogen 1.5 mL tube Magnetic Rack







Day 1
Jan 14, 2023
1. Buffer preparation: Make CUT&RUN buffers fresh the day of use.
1) Add 1.8 mL Pre-Wash Buffer per sample to a 50 mL conical tube labeled "Wash Buffer". 
2) Dissolve 1 protease inhibitor tablet (Roche) in 2 mL water (25X stock). Add 72 uL per sample to the Wash Buffer. Store remaining 25X stock for 12 weeks at -20°C.
3) Dilute 1M Spermidine 1:2,000 in the Wash Buffer. Store final buffer at RT.
4) Transfer 1.4 mL of Wash Buffer per sample into a new 50 mL conical tube labeled "Cell Perm Buffer". Add 5% Digitonin (1:500 dilution).
5) Transfer 100 uL per sample of Cell Permeabilization Buffer into a new 50 mL tube labeled "Antibody Buffer". Add 0.5 M EDTA (1:250 dilution). Store final buffer on ice.
6) Store the remaining Cell Perm Buffer at 4°C overnight (for Day 2 use).



A.Wash buffer: Leave at RT for use on Day 1
Per sample: 1.8 mL Pre-Wash Buffer, 72 uL Protease inhibitor (1X final), 0.9 uL Spermidine (0.5 mM final)
B.Cell Permeabilization Buffer: Store at 4°C overnight for use on Day 2
Per sample: 1.4 mL Wash Buffer, 2.8 uL Digitonin (0.01% final)
C.Antibody Buffer: Set on ice for use on Day 1
Per sample: 100 ul Cell Perm. Buffer, 0.4 uL EDTA (2 mM final)


2. Bead activation 
1) Gently resuspend theConA Beadsby pipetting.Transfer 11 uL/sample to a 1.5 mL tube for batch processing.
6 samples: Total 66 ul 
2) Place the tube on a magnet until slurry clears and pipette to remove sup.
3) To avoid drying the beads, immediately add 100 uL/sample coldBead Activation Buffer. Pipette gently to mix.
4) Place the tube on a magnet until slurry clears and pipette to remove sup. Repeat previous step for total of two washes.
5) Resuspend beads in 11 uL/sample coldBead Activation Buffer.
6) Aliquot 10 uL/sample of activated bead slurry into separate 8-strip tubes. Keep on ice until needed.
7) Harvest 0.5 million cells/sample in 1.5 mL tube. Centrifuge at 600 x g, 3 min at RT. Decant or pipette culture media sup.
a) 0.5 M: FLAG, b) 1.0 M: FLAG, c) 0.5 M: V5, d) 1.0 M: V5, e) 0.5 M: IgG negative control, f) 0.5 M: H3K4me3 positive control
8) Resuspend cells in 100 uL/sample RTWash Buffer. Pipette to thoroughly resuspend.Centrifuge at 600 x g, 3 min at RT. Decant or pipette sup.
9) Repeat previous step for total of two washes.
10) Resuspend cells in 105 uL/sample in RTWash Bufferand thoroughly pipette to mix. Aliquot 100 uL washed cells to each 8-strip tube containing 10 uL of activated beads. Gently vortex and/or pipette until evenly resuspended.
11) Incubate cell-bead slurry on benchtop for 10 min at RT to adsorb cells to beads.


3. Antibody binding
12) If using a multi-channel pipette (recommended) place a multi-channel reagent reservoir on ice.
13) Place the 8-strip tubes on an 8-strip magnet (high volume setting) until slurry clears. Pipette to remove sup, taking care to avoid disturbing beads with tip.
14) Immediately add 50 uL coldAntibody Bufferto each sample and gently vortex and/or pipette mix to prevent beads from drying.
15) Add 2 uL CUTANA H3K4 MetStat Spike-in Control dNucs to the cell samples designated for the positive (H3K4me3) and negative (IgG) control antibodies.
16) Add 0.5 ug antibody to each sample and gently vortex.
*** Control antibodies included in this kit are provided at 0.5 mg/mL, therefore add 1.0 uL per sample.
FLAG antibody: 1 mg/mL -> 0.5 uL
V5 antibody: 1 mg/mL -> 0.5 uL
IgG, H3K4me3 antibodies: 1.0 uL
17) Incubate 8-strip tubes on nutator (capped ends elevated) overnight at 4°C
18) Store theCell Permeabilization Bufferat 4°C overnight for use on Day 2.




Day 2 
Jan 15, 2023
4. Antibody binding, continued
19)If using a multi-channel pipette (recommended) place a multi-channel reagent reservoir on ice.Fill withCell Permeabilization Buffer.
20) Place the 8-strip tubes on magnet until slurry clears. Pipette to remove sup.
21)While beads are on magnet, add 200 uL coldCell permeabilization Bufferdirectly onto beads. Pipette to remove sup.
22) Repeat previous step for total of two washes, without removing 8-strip tubes from the magnet.
23) Add 50 uL coldCell Permeabilization Bufferto each sample. Gently vortex and/or disperse clumps by thorough pipetting.
Permeabilization: the puncturing of the cell membrane


5. Binding of pAG-MNase 
pAG-MNase enzyme is a fusion of protein A and protein G to micrococcal nuclease.
24) Add 2.5 uL pAG-MNase (20X stock) to each sample. Gently vortex/pipette mix.
25) Incubate samples for 10 min at RT. Return 8-strip tube to magnet. Remove sup.
26)While beads are on magnet,add 200 uL cold Cell permeabilization Buffer directly onto beads. Pipette to remove sup.
27)Repeat previous step for total of two washes, without removing 8-strip tubes from the magnet.
28) Remove 8-strip tubes from the magnet. Add 50 uL coldCell permeabilization Bufferto each sample. Gently vortex and/or disperse clumps by thorough pipetting.


6. Targeted chromatin digestion and release
29) Place 8-strip tubes on ice. Add 1 uL100 mM Calcium Chlorideto each sample and gently vortex. Ensure efficient digestion by making sure beads are thoroughly resuspended. Gently pipette with a P200 if needed.
30) Incubate 8-strip tubes on nutator for 2 hours at 4°C
31) Add 33 uL Stop Buffer to each sample. Gently vortex to mix.
32) Prior to first use, reconstituteE.coliSpike-in DNA in 200 uL DNase free water.
33) Add 1 uL (0.5 ng) Spike-in DNA to each sample. Gently vortex to mix.
34) Incubate 8-strip tubes for 10 min at 37°C in a thermocycler. (Setting: Incubate mode)
35) Quick spin in benchtop microfuge.
36) Place 8-strip tubes on a magnet stand until slurry clears. Transfer sups containing CUT&RUN enriched DNA to 1.5 mL tubes and discard ConA Beads.


7. DNA purification
37) Add 420 uL DNA Binding Buffer to each sample. Mix well by vortexing.
38) For every sample, place a DNA Cleanup Column into a DNA Collection Tube. Load each sample onto a column and label the top.
39) Centrifuge at 16,000 x g, 30 sec, RT. Discard the flow-through. Place the column back into the collection tube.
40) Prior to first use, add 20 mL > 95% ethanol toDNA Wash Buffer.



8. DNA purification, continued
41) Add 200 uL DNA Wash Buffer to each sample column.
42)Centrifuge at 16,000 x g, 30 sec, RT. Discard the flow-through. Place the column back into the collection tube.
43) Repeat for a total of two washes.
44)Discard the flow-through.Centrifuge one additional time at16,000 x g, 30 sec to completely dry the column (Empty spin).

45) Carefully remove the column from the collection tube, ensuring it does not come into contact with the flow-through. Transfer column to a clean pre-labeled 1.5 mL microfuge tube.
46) Elute DNA by adding 12 uLDNA Elution Buffer, taking care to ensure the buffer is added to the center of the column rather than the wall. Tap the column + microfuge collection tube on the benchtop to ensure all droplets are absorbed onto the resin.
*** 12 uL is recommended, however DNA can be eluted in 6-20 uL volumes depending on anticipated yield and desired final concentration. Larger elution volumes, longer incubation times, and/or multiple rounds of elution may improve DNA yield. However, sample concentration will be reduced with larger total elution volume.
47) Let sit 5 minutes, then centrifuge at16,000 x g, 1 min, RT.
48) Vortex eluted material and use 1 uL to quantify the CUT&RUN-enriched DNA using the Qubit fluorometer as per the manufacturer's instructions. See Quality Control Checks section for typical DNA yields.
49) CUT&RUN DNA can be stored at -20°C for future processing.


Measure the DNA concentration using Nanodrop
Blank: DNA Elution Buffer 1ul






Day 3
Jan , 2023
9. NGS library preparation