TITLE: CUT&amp;RUN for nuclei using the CUTANA™ ChIC/CUT&amp;RUN Kit
AUTHORS: Eric RA Pederson

[DESCRIPTION]
Modified CUTANA™ ChIC/CUT&RUN Kit protocol using nuclei from brain tissue.

[BEFORE_START]
The pre-experimentation steps are important 
Also it is important that all the primers have been ordered and reconstituted beforehand.

[GUIDELINES]
Taken from the Cutana ChIC/CUT&RUN Kit (https://www.epicypher.com/products/epigenetics-reagents-and-assays/cutana-chic-cut-and-run-kit) for more information.

[STEPS]
SECTION: pre-experimentation
1. All steps should be performed on ice or at 4 °C. 
SECTION: pre-experimentation
2. Pre-chill all Dounces and pestles to 4 °C in a fridge. Or just leave on ice for a while.

SECTION: pre-experimentation
4. Prepare buffers. 
i) Homogenization buffer
ii) 25X protease inhibitor
iii) wash buffer (see step 5)
iv) CP buffer (see step 5)
v) antibody buffer (see step 5)
vi) Nuclear Buffer (see step 26)

SECTION: DAY 1 - Section I - Buffer Prep
5.  
                  		 			# of Samples 		 		 			1X 		 		 			2X 		 		 			3X 		 		 			4X 		 		 			5X 		 		 			6X 		 		 			8X 		 	  		 			Wash Buffer – store at 			RT for use on Day 1 		 	  		 			Pre-wash buffer 		 		 			1.8 ml 		 		 			3.6 ml 		 		 			5.4 ml 		 		 			7.2 ml 		 		 			9 ml 		 		 			10.8 ml 		 		 			14.4 ml 		 	  		 			W1 Additive 		 		 			0.18 ul 		 		 			0.36 ul 		 		 			0.54 ul 		 		 			0.72 		 		 			0.90 ul 		 		 			1.08 ul 		 		 			1.44 ul 		 	  		 			25X Protease inhibitor 		 		 			72 ul 		 		 			144 ul 		 		 			216 ul 		 		 			288 ul 		 		 			360 ul 		 		 			432 ul 		 		 			576 ul 		 	  		 			Cell Permeabilization 			buffer – store at 4° for use on Day 2 		 	  		 			Wash Buffer 		 		 			1.4 ml 		 		 			2.8 ml 		 		 			4.2 ml 		 		 			5.6 ml 		 		 			7 ml 		 		 			8.4 ml 		 		 			11.2 ml 		 	  		 			CP2 Additive 		 		 			2.8 ul 		 		 			5.6 ul 		 		 			8.4 ul 		 		 			11.2 ul 		 		 			14 ul 		 		 			16.8 ul 		 		 			22.4 ul 		 	  		 			Antibody Buffer – store 			on ice for use on Day 1 		 	  		 			Cell Permeabilization 			buffer 		 		 			100 ul 		 		 			200 ul 		 		 			300 ul 		 		 			400 ul 		 		 			500 ul 		 		 			600 ul 		 		 			800 ul 		 	  		 			AB3 Additive 		 		 			0.4 ul 		 		 			0.8 ul 		 		 			0.16 ul 		 		 			0.32 ul 		 		 			0.64 ul 		 		 			0.128 ul 		 		 			3.2 ul 		 	   
Wash buffer - store at RT for use on Day 1
CP buffer - store at 4° for use on Day 2
Antibody buffer - store on ice for use on Day 1
SECTION: DAY 1 - Section I - Buffer Prep
6. Example of making solutions:

Add 1.8 mL of Pre-Wash Buffer per sample to a single conical tube labeled Wash Buffer
Add 72 µL  μL of 25X protease inhibitor stock per sample to the Wash Buffer
Add 0.9 µL  ul of 1M Spermindine per sample to the Wash Buffer

Room temperature

SECTION: DAY 1 - Section I - Buffer Prep
7. Transfer 1.4 mL of Wash Buffer per sample into a new conical tube labelled Cell Permeabilization Buffer
Add 2.8 µL of 5% digitonin  to CP buffer

Store the remaining Cell Permeabilization Buffer at 4 °C   (for Day 2 use). 


SECTION: DAY 1 - Section I - Buffer Prep
8. Transfer 100 µL per sample of Cell Permeabilization Buffer into a new tube labelled “Antibody Buffer”

Add 0.4 µL 0.5 Molarity (m) EDTA per sample

Store final buffer on ice.
SECTION: Section II - Bead Activation
9. Gently resuspend the ConA Beads by pipetting. 

Transfer 11 µL/sample to a 1.5 mL tube for batch processing. 

*NOTE: Batch processing at this step is recommended to improve sample handling.
 If a 1.5 mL tube magnet is not available, the beads can be processed individually (10 μL/ sample) in the provided  8-strip PCR tubes using a compatible 8-strip magnet. 

SECTION: Section II - Bead Activation
10. Place the tube on a magnet until slurry clears and pipette to remove sup. 

*IMPORTANT: For all steps involving magnetic racks, take care to avoid disturbing the immobilized beads with pipette tips. 

SECTION: Section II - Bead Activation
11. To avoid drying the beads, immediately add 100 µL/sample cold Bead Activation Buffer. 

Pipette gently to mix. 

SECTION: Section II - Bead Activation
12. Place the tube on a magnet until slurry clears and pipette to remove sup. 	

Repeat previous step for total of two washes. 

SECTION: Section II - Bead Activation
13. Resuspend beads in 11 µL/sample cold Bead Activation Buffer.

*NOTE: If not batch processing, use 10 μL/sample at this step. Proceed directly to Section III. 

SECTION: Section II - Bead Activation
14. For each experimental condition, aliquot 10 µL/sample of activated bead slurry into separate 8-strip tubes. 

Keep on ice until needed. 	

SECTION: Section III - Binding cells to activated beads
25.  
      		 			Materials needed 		 		 			Recipe 		 	  		 			Nuclear Extraction buffer 		 		 			below 		 	  		 			Protease inhibitor 		 		 			Roche #11873580001 		 	  		 			Wash Buffer (W1) additive 		 		 			Cutana 		 	  		 			PBS 		 		 			Doesn’t matter 		 	  		 			0.4% trypan blue 		 		 			Invitrogen #T10282 		 	  		 			Cell counter 		 		 			Cell lab 		 	 
 
SECTION: Section III - Binding cells to activated beads
26. Prepare 410 µL  / sample (+ extra dead volume) Nuclear Extraction Buffer fresh the day of use. Sterile Filter.

SECTION: Section III - Binding cells to activated beads
27. Add 1:10,000 dilution of 1 Molarity (m) Spermindine and 1X Protease Inhibitor to the Nuclear Extraction Buffer. Place final buffer on ice.  

200/10000 = 0.2 ul
SECTION: Section III - Binding cells to activated beads
28. Resuspend cells in 100 µL per sample 1X PBS. 

Set aside 10 µL cells for future analysis by Trypan blue staining (intact cell control).

Centrifuge for 600 rpm, 3 min, 4 °C . Remove and discard supernatant

*NOTE:For all steps, the ratio of buffer volumes; cells scales linearly. For example, use 1 mL buffer for 5 x 10^6 cells.

SECTION: Section III - Binding cells to activated beads
29. Resuspend cells in 200 µL per sample cold Nuclear Extraction Buffer.

SECTION: Section III - Binding cells to activated beads
30. Incubate 0 °C  (ice)

SECTION: Section III - Binding cells to activated beads
31. Centrifuge for 600 rpm, 3 min, 4 °C 

Remove and discard sup. 

The pellet should change in appearance from sticky, pale yellow (cells) to white and fluffy (nuclei).
SECTION: Section III - Binding cells to activated beads
32. Add 10 µL Trypan blue to the intact cell control (Step 4) and the isolated nuclei (previous step). 

SECTION: Section III - Binding cells to activated beads
33. Load onto hemacytometer slide and examine under brightfield/phase microscope to determine whether nuclei have been efficiently isolated (Figure 12). 
SECTION: Section III - Binding cells to activated beads
34. To cryopreserve nuclei, slowly freeze samples in isopropanol-filled chiller in -80 °C freezer. 

SECTION: Section III - Binding cells to activated beads
35. When ready to use samples for CUT&RUN, thaw nuclei quickly by placing on 37 °C block. 
Centrifuge down and reconstitute
SECTION: Section III - Binding cells to activated beads
36. Proceed to CUT&RUN ConA Bead conjugation step (Experimental Protocol, Step 10).
SECTION: Section III - Binding cells to activated beads
37. Resuspend cells in 105 µL /sample in RT Wash Buffer. Pipette to thoroughly resuspend. 

Aliquot 100 µL washed cells to each 8-strip tube containing  10 µL of activated beads. Gently vortex or pipette to mix.

*NOTE: Beads are prone to clumping. If your beads are clumped, continue to vortex and/or pipette mix to ensure even resuspension. 
SECTION: Section III - Binding cells to activated beads
38. Incubate cell-bead slurry on the platform rocker for 10 min at Room temperature to adsorb cells to beads. 


*NOTE: Count cells by Trypan staining prior to incubation with ConA beads. After incubation with ConA beads, check the sup to ensure most cells have adsorbed to the beads. 
SECTION: Section IV - ANTIBODY BINDING
45. If using a multi-channel pipette (recommended) place a multi-channel reagent reservoir on ice. 

*IMPORTANT: If processing > 8 samples (more than 1 x 8-strip tube), for subsequent wash steps remove & replace sups for a single strip before processing the next strip. This avoids bead dry out during wash steps. 

*NOTE: Multi-channel pipetting is highly recommended through the rest of the experiment. This helps to avoid bead dry out, improves yield, and increases experimental throughput. 
SECTION: Section IV - ANTIBODY BINDING
46. Place the 8-strip tubes on an 8-strip tube magnet (high volume setting) until slurry clears. 

Pipette to remove sup, taking care to avoid disturbing the immobilized beads with pipette tip. 

SECTION: Section IV - ANTIBODY BINDING
47. Immediately add 50 µL cold Antibody Buffer to each sample and gently vortex and/or pipette mix to prevent beads from drying. 

SECTION: Section IV - ANTIBODY BINDING
48. (Optional)
Add 2 µL SNAP-CUTANA K-MetStat Panel per 500,000 cells. 
The samples designated for the positive (H3K4me3) and negative (IgG) control antibodies. 
SECTION: Section IV - ANTIBODY BINDING
49. Add 0.5 µg antibody to each sample and gently vortex.

SECTION: Section IV - ANTIBODY BINDING
50. Incubate 8-strip tubes on rotator   at 4 °C. 

SECTION: DAY 2 - Section IV - Antibody binding
51. If using a multi-channel pipette (recommended) place a multi-channel reagent reservoir on ice. 

Fill with Cell Permeabilization Buffer. 


*IMPORTANT: If processing > 8 samples (more than 1 x 8-strip tube), for subsequent wash steps remove & replace sups for a single strip before processing the next strip. This avoids bead dry out during wash steps. 
SECTION: DAY 2 - Section IV - Antibody binding
52. Place the 8-strip tubes on magnet until slurry clears. Pipette to remove sup. 
SECTION: DAY 2 - Section IV - Antibody binding
53. *While beads are on magnet*, add 200 µL cold Cell Permeabilization Buffer directly onto beads. 

Pipette to remove supernatant
SECTION: DAY 2 - Section IV - Antibody binding
54. Repeat previous step for total of two washes, without removing 8-strip tubes from the magnet. 
SECTION: DAY 2 - Section IV - Antibody binding
55. Add 50 µL cold Cell Permeabilization Buffer to each sample. 

Gently vortex and/or disperse clumps by thorough pipetting. 
SECTION: Section V - BINDING OF PAG-MNASE
56. Add 2.5 µL pAG-MNase (20x stock) to each sample. 

Gently vortex/pipette mix. 

*NOTE: To evenly distribute enzyme across cells/nuclei, ensure beads are thoroughly 
resuspended by gentle pipetting with a P200. 
SECTION: Section V - BINDING OF PAG-MNASE
57. Incubate samples for 10 min at Room temperature on the platform shaker

Return 8-strip tube to magnet. 

Remove sup. 	

SECTION: Section V - BINDING OF PAG-MNASE
58. *While beads are on magnet*, add 200 µL cold Cell Permeabilization Buffer directly onto beads. 

Pipette to remove sup. 

SECTION: Section V - BINDING OF PAG-MNASE
59. Repeat previous step for total of two washes without removing 8-strip tubes from the magnet.
SECTION: Section V - BINDING OF PAG-MNASE
60. Remove 8-strip tubes from the magnet. 

Add 50 µL cold Cell Permeabilization Buffer to each sample. 

Gently vortex and disperse clumps by pipetting. 

Cover/ put away Cell Permeabilization Buffer for later use. 

SECTION: Section VI - TARGETED CHROMATIN DIGESTION AND RELEASE
61. Place 8-strip tubes on ice. 

Add 3 µL Calcium Chloride to each sample and gently vortex. 

Ensure efficient digestion by making sure beads are thoroughly resuspended. 

Gently pipette with a P200 if needed.
SECTION: Section VI - TARGETED CHROMATIN DIGESTION AND RELEASE
62. Incubate 8-strip tubes on rotator for 120 min at 4 °C.

SECTION: Section VI - TARGETED CHROMATIN DIGESTION AND RELEASE
63. Add 33 µL Stop Buffer to each sample. 

Gently vortex to mix.
SECTION: Section VI - TARGETED CHROMATIN DIGESTION AND RELEASE
64. Prior to first use, reconstitute E. coli Spike-in DNA in 200 µL DNase free water. 

*IMPORTANT: Lyophilized DNA pellet is invisible to the eye. 

Prior to opening, pellet DNA by quick spin in a benchtop microfuge. 

After reconstitution, vortex tube on all sides to ensure complete resuspension. 
SECTION: Section VI - TARGETED CHROMATIN DIGESTION AND RELEASE
65. Add 1-2 µL Spike-in DNA to each sample. Gently vortex to mix. 


*NOTE: In general, aim for Spike-in DNA to comprise 0.5 – 5% (ideally closest to 1%) of total read counts in the sequencing data. 

Therefore, while 0.5 ng is a good starting amount for both high (e.g. H3K27me3) and low (e.g. H3K4me3) abundance targets, this may need to be adjusted higher or lower depending on the antibody used, target of interest, total DNA yield, and sequencing results. 
SECTION: Section VI - TARGETED CHROMATIN DIGESTION AND RELEASE
66. Incubate 8-strip tubes for 10 minat 37 °C in a thermocycler. 

SECTION: Section VI - TARGETED CHROMATIN DIGESTION AND RELEASE
67. Quick spin in benchtop microfuge. 

SECTION: Section VI - TARGETED CHROMATIN DIGESTION AND RELEASE
68. 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. 

SECTION: Section VII - DNA PURIFICATION
69. Add 420 µL DNA Binding Buffer to each sample. 

Mix well by vortexing. 

SECTION: Section VII - DNA PURIFICATION
70. For every sample, place a DNA Cleanup Column into a DNA Collection Tube. 

Load each sample onto a column and label the top. 

SECTION: Section VII - DNA PURIFICATION
71. Centrifuge for 16.000 x g, 30 s Room temperature 

 Discard the flow-through. 

Place the collection tube back on to the column. 


*NOTE: A vacuum manifold can be used in place of centrifugation. 

For each step, add the indicated buffer, turn the vacuum on, and allow the solution to pass through the column before turning the vacuum off. 
SECTION: Section VII - DNA PURIFICATION
72. Prior to first use, add 20 mL ≥ 95% ethanol to DNA Wash Buffer. 

SECTION: Section VII (continued)
73. SECTION VII: DNA PURIFICATION (~30 MIN), CONTINUED 
SECTION: Section VII (continued)
74. Add 200 µL DNA Wash Buffer to each sample column

SECTION: Section VII (continued)
75. Centrifuge for16.000 x g, 30 s  , Room temperature

Discard the flow-through. 

Place the collection tube back on to the column. 

SECTION: Section VII (continued)
76. Repeat for a total of two washes. 

SECTION: Section VII (continued)
77. Discard the flow-through. 

Centrifuge one additional time for 16.000 x g, 30 s to completely dry the column.

SECTION: Section VII (continued)
78. Transfer column to a clean pre-labeled 1.5 mL lobind tube, ensuring the column does not come into contact with the flow-through. 

SECTION: Section VII (continued)
79. Elute DNA by adding 12 µL DNA Elution Buffer, taking care to ensure the buffer is added to the center of the column rather than the wall. 

Tap the column + collection tube on the benchtop to ensure all droplets are absorbed onto the resin. 

*NOTE: 12 μL is recommended, however DNA can be eluted in 6 – 20 μL 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. 
SECTION: Section VII (continued)
80. Let sit5 min, then centrifuge for 16.000 x g, 1 min, RT. 

SECTION: Section VII (continued)
81. Vortex eluted material and use 1 µL 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. 
SECTION: Section VII (continued)
82. CUT&RUN DNA can be stored at -20 °C for future processing.

SECTION: Section VIII - Library Preparation
83. 1) End Prep


Starting Material: 500 pg–1 µg fragmented DNA. We recommend that DNA be sheared in 1X TE

If the DNA volume post shearing is less than 50 µL, add 1X TE to a final volume of 50 µL 

Alternatively, samples can be diluted with 10 millimolar (mM) Tris-HCl, pH 8.0 or 0.1X TE. 

SECTION: Section VIII - Library Preparation
84. NEBNext End Prep 

Add the following components to a sterile nuclease-free tube:


 

SECTION: Section VIII - Library Preparation
85. Set a 100 µL or 200 µL pipette to 50 µL and then pipette the entire volume up and down at least 10 times to mix thoroughly. 	

Perform a quick spin to collect all liquid from the sides of the tube. 

Note: It is important to mix well. The presence of a small amount of bubbles will not interfere with performance. 

SECTION: Section VIII - Library Preparation
86. Place in a thermocycler, with the heated lid set to ≥ 75 °C, and run the following program: 


 


If necessary, samples can be stored at –20°C; however, a slight loss in yield (~20%) may be observed. We recommend continuing with adaptor ligation before stopping. 
SECTION: Section VIII - Library Preparation
87. Serapure purification


- Add 1.8X Speed beads to samples (108 µL beads to 60 µL reaction)
- Flick to mix
- Incubate for 10 min at room temperature
- Use the magnet to collect the DNA-beads
- Discard supernatant
- Wash with 70% ethanol
- Dry at 37 °C for 3 min
- Elute with 20 µL TE buffer
- Incubate for 5 min at room temperature
- Use the magnet to collect the DNA-beads
- Collect supernatant into a new tube
SECTION: Section VIII - Library Preparation
88. Universal Adapter ligation


Add the following components directly to the End Prep Reaction Mixture: 


 

  

SECTION: Section VIII - Library Preparation
89. Incubate the reaction for 10 min at room temperature.

SECTION: Section VIII - Library Preparation
90. Serapure purification


- Add 0.8X Speed beads to samples (20 µL Speed beads to 25 µL reaction)
- Flick to mix
- Incubate for 10 min at room temperature
- Use the magnet to collect the DNA-beads
- Discard supernatant
- Wash with 70% ethanol
- Dry at 37 °C for 3 min
- Elute with 21 µL TE buffer
- Incubate for 5 min at room temperature
- Use the magnet to collect the DNA-beads
- Collect supernatant into a new tube
SECTION: Section VIII - Library Preparation
91. PCR and primer indexing


 

SECTION: Section VIII - Library Preparation
92. PCR and primer indexing according to the following cycling parameters: 


 

SECTION: Section VIII - Library Preparation
93. Serapure purification

- Add 1.0X Speed beads to samples (50 µL Speed beads to 50 µL reaction)
- Flick to mix
- Incubate for 10 min at room temperature
- Use the magnet to collect the DNA-beads
- Discard supernatant
- Wash with 70% ethanol
- Dry at 37 °C for 3 min
- Elute with 20 µL TE buffer
- Incubate for 5 min at room temperature
- Use the magnet to collect the DNA-beads
- Collect supernatant into a new tube
SECTION: Section VIII - Library Preparation
94. Qubit and Tapestation (D1000) analysis
SECTION: pre-experimentation
3. Pre-chill all tubes.

For each sample you are processing, you will need: 
(i) One 2 mL    per sample
(ii) Three   per sample
(iii) one PCR tube per sample 
(iv) One   for filtration step per sample
SECTION: Tissue preparation and Sample Preparation: Nuclei
15. In the most sterile way possible, cut a small piece of tissue, around 50 mg, and leave it in the petri dish with a marking on the lid, in the dry ice. Weigh it and cut again if needed.

Make sure to use the ethanol to clean everything and be careful not to cut yourself.
SECTION: Tissue preparation and Sample Preparation: Nuclei
16. Add 2 mL of 1X HB buffer into the dounce, which is sitting in the ice.

SECTION: Tissue preparation and Sample Preparation: Nuclei
17. Place 20 mg frozen tissue into a pre-chilled 2 mL Dounce containing 2 mL cold 1x HB and let thaw for 5 min.

SECTION: Tissue preparation and Sample Preparation: Nuclei
18. Dounce with “A” loose pestle until resistance goes away (~10 strokes).

Put the A pestle into the beaker of water
SECTION: Tissue preparation and Sample Preparation: Nuclei
19. Dounce with “B” tight pestle for 20 strokes.

Put the B pestle into the beaker of water
SECTION: Tissue preparation and Sample Preparation: Nuclei
20. Pour everything from the dounce into a 30 um MACS smartstrainer which is sitting on top of a labelled 50 mL falcon tube sitting in ice.
 

SECTION: Tissue preparation and Sample Preparation: Nuclei
21. Let it drip through for15 min 

SECTION: Tissue preparation and Sample Preparation: Nuclei
22. Transfer to a labelled lobind eppendorf tube, already cold from sitting in ice.
SECTION: Tissue preparation and Sample Preparation: Nuclei
23. To pellet the nuclei, centrifuge 900 rpm, 10 min, 4 °C 

SECTION: Tissue preparation and Sample Preparation: Nuclei
23.1. Transfer the supernatant to a new tube without disturbing the pellet

Repeat the centrifugation
SECTION: Tissue preparation and Sample Preparation: Nuclei
24. Discard supernatant
SECTION: pre-experimentation
4.1. Homogenization buffer (HB); 
 
    		10mM 		Tris pH 8.0 	  		250mM 		sucrose 	  		25mM 		KCl 	  		5mM 		MgCl2 	  		0.10% 		Triton X-100  	  		0.50% 		RNasin Plus RNase inhibitor Promega Catalog #N2611   	  		0.20% 		1X protease Inhibitor Promega Catalog #G6521  	  		0.1mM 		DTT 	 
 
   
   

SECTION: pre-experimentation
4.2. ii) 25X protease inhibitor

1 protease inhibtor tablet (Roche) in 2 mL water (25X stock)
Store remaining 25X stock for 12 weeks at -20 °C 
  

SECTION: Sample Preparation: Cross-Linking (Optional)
40. Labile targets or highly transient chromatin binding proteins may be improved by cross-linking
SECTION: Sample Preparation: Cross-Linking (Optional)
41. Transfer 500,000 cells or amount that you wish to use into a 1.5 mL lobind tube

SECTION: Sample Preparation: Cross-Linking (Optional)
42. Add formaldehyde directly to culture to achieve desired final concentration of formaldehyde.
(recommended 0.1%-1%) 
SECTION: Sample Preparation: Cross-Linking (Optional)
42.1. I use 16% formaldehyde in 200 µL of nuclear extraction buffer, which requires 13.75 µL of formaldehyde.

SECTION: Sample Preparation: Cross-Linking (Optional)
43. Incubate Room temperature  on 


 

SECTION: Sample Preparation: Cross-Linking (Optional)
44. Quench the fixation by adding 2.5 Molarity (m) glycine to a final concentration of 125 millimolar (mM)


In this case it would be 11 µL

SECTION: Sample Preparation: Cross-Linking (Optional)
44.1.  600 rpm, 3 min, 4 °C 
SECTION: Sample Preparation: Cross-Linking (Optional)
44.2. Discard supernatant
39.                                                                             
SECTION: Section III - Binding cells to activated beads
34.1. It might be a good idea to add 300 µL of freezing medium to each tube for saving

 
      		Hepes pH 7.5  		10 mM  		1 ml  		1 M stock  	  		MgCl2 		2 mM 		200 ul   		1 M stock  	  		KCl 		25 mM  		800 ul 		3 M stock  	  		H20 		 		98 ml  		 	 
 
Mix 15 mL of buffer N containing 5 µL Halt protease inhibitor cocktail,  1 millimolar (mM) DTT and 1 millimolar (mM) PMSF with 35 mL of pure glycerol 

  
  
  

SECTION: Section III - Binding cells to activated beads
38.1. I have been moving the tube to ice for 50 min as an extra time incubation

SECTION: Section VI - TARGETED CHROMATIN DIGESTION AND RELEASE
66.1. (Optional - if Cross-Linking extra methodology only)

Place 8-strip tubes on magnet stand until slurry clears.

Transfer supernatants containing DNA to new 8-strip tubes
SECTION: Section VI - TARGETED CHROMATIN DIGESTION AND RELEASE
66.2. Reverse cross-links by adding 0.8 µL 10% SDS and 1 µL of 20 µg/µL Proteinase K to each reaction.

Mix by vortexing
SECTION: Section VI - TARGETED CHROMATIN DIGESTION AND RELEASE
66.3.   55 °C 
in a thermocycler
SECTION: Section VIII - Library Preparation
88.1. Or one can skip the purification and run the ligation suggested in the NEB protocol

 
    		End Prep Reaction Mixture  		60 ul 	  		NEBNext Ligation Enhancer  		2.5 μl 	  		NEBNext Ligation Master Mix  		30 ul 	  		NEBNext Adaptor  		1 μl 	  		Total volume  		93.5 μl