TITLE: Micro-C protocol for frozen tissue using the Dovetail® Micro-C Kit
AUTHORS: Eric RA Pederson

[DESCRIPTION]
micro-C protocol from Dovetail for the use on frozen tissue, in this case 

[BEFORE_START]
Complete everything in the pre-epxerimentation section

[GUIDELINES]
The amount of mononuclease content must be between 30-70%

[STEPS]
SECTION: pre-experimentation
1. All steps should be performed on ice or at 4 °C. 
SECTION: pre-experimentation
2. Autoclave cryogrinder pieces wrapped in foil
SECTION: pre-experimentation
3. pre-cool the cryogrinder pieces
SECTION: pre-Step 1
5. Stage 1: Crosslinking, Digestion and Lysis 

As you prepare for Stage 1, keep the following in mind: 

Sample preparation takes ~ 1.5 hours.

SECTION: Step 1
12. Weigh out 20 mg of frozen tissue sample. 

If using muscle tissue, weigh out 50 mg.
SECTION: Step 1
13. Grind the sample to a fine powder (flour-like consistency) with a mortar and pestle in liquid nitrogen. 

 

Ensure the sample is kept frozen while grinding.
SECTION: Step 1
14. Transfer the ground tissue sample to a 1.5 mL tube containing: 1mL 1XPBS, 10μL0.3 DMSG  

SECTION: Step 1
15. Rotate the tube for 10 minutes at room temperature. 

Cells should not settle.
SECTION: Step 1
16. Add 62.5 μL 16% formaldehyde.

  

SECTION: Step 1
17. Rotate the tube for 10 minutes at room temperature. 

Cells should not settle.
SECTION: Step 1
18. Spin the tube at 3,000 x g for 5 minutes. 

Carefully remove and discard the supernatant. Use caution, the pellet might be loose.
SECTION: Step 1
19. Wash the pellet with a total of 1 mL 1X Wash buffer: first add 200 μL of Wash Buffer and pipette up and down to break up clumps then add the remaining 800 μL. 

Pipette up and down to fully resuspend the pellet.
SECTION: Step 1
20. Spin the tube at 3,000 x g for 5 minutes. 

Carefully remove and discard the supernatant.
SECTION: Step 1
21. Repeat steps 8 and 9 once, for a total of 2 washes.
SECTION: Step 1
22. After removing the second wash, resuspend the pellet in 1 mL 1X Wash Buffer. 

Pipette up and down to fully mix.
SECTION: Step 1
23. Using a 1 mL syringe, gently push the 1 mL resuspended sample through a 200 μm filter into a new 5 mL tube.

  

SECTION: Step 1
24. If the filter clogs, replace with a new 200 μm filter and continue until all of the sample has been filtered.
SECTION: Step 1
25. Gently pass an additional 1 mL of 1X Wash Buffer though the 200 μm filter into the 5 mL tube.

 Your tube should now contain a total volume of ~2 mL.
SECTION: Step 1
26. Using the same syringe but changing to a 50 μm filter, re-filter the 2 mL sample into a new 5 mL tube.
SECTION: Step 1
27. Gently pass an additional 1 mL of 1X Wash Buffer though the 50 μm filter into the 5 mL tube. 

Your tube should now contain a total volume of ~3 mL.
SECTION: Step 1
28. Spin the tube at 3,000 x g for 5 minutes. 

Carefully remove and discard the supernatant.
SECTION: Step 1
29. Resuspend the pellet in 50 μL 1X Nuclease Digest Buffer 
SECTION: Step 1
30. Add 0.5 μL of MNase Enzyme Mix.

 Pipette up and down to fully mix.
SECTION: Step 1
31. Incubate the tube at 22° for exactly 15 minutes in an agitating thermal mixer set at 1,250 rpm. 

If you are working with a large number of samples, stagger the start of the digestion for each sample by 20 seconds then stop after corresponding 15 minutes
SECTION: Step 1
32. Stop the reaction by adding 5 μL of 0.5 M EGTA. 

Pipette up and down to fully mix.
SECTION: Step 1
33. Add 3 μL of 20% SDS to lyse the cells. 

Pipette up and down to fully mix.
SECTION: Step 1
34. Incubate at 22° for 5 minutes in an agitating thermal mixer set at 1,250 rpm.
SECTION: Step 1
35. Continue to Stage 2: Lysate QC, page 13.
SECTION: pre-step 2
36. Stage 2: Lysate QC 
As you prepare for Stage 2, keep the following in mind: 
 The Lysate QC stage takes ~ 2 hours. 

This stage has 2 objectives: 

Quantify the lysate to determine the volume of lysate to use in Stage 3. 

Confirm that the chromatin was properly digested. 

Recommended kit; D5000 HS (Tapestation)

SECTION: Step 2
40. Transfer 2.5 μL of the lysate to a new 1.5 mL tube labelled QC.
SECTION: Step 2
41. NOTE: Store the remainder of the lysate at -80°. 

This is the lysate you will be using in Stage 3.
SECTION: Step 2
42. Digest Buffer (50 μL), mix the following components:
SECTION: Step 2
42.1.  
              		 			Reagent 		 		 			1X 		 		 			10% 			extra 		 		 			3X 		 		 			5X 		 		 			8X 		 	  		 			UltraPure 			water 		 		 			45 			ul 		 		 			49 			ul 		 		 			147 ul 		 		 			245 ul 		 		 			392 ul 		 	  		 			10x 			Crosslink Reversal 			buffer 		 		 			5 ul 		 		 			5.5 ul 		 		 			16.5 ul 		 		 			27.5 ul 		 		 			44 ul 		 	  		 			Proteinase 			K 		 		 			1.5 ul 		 		 			1.65 			ul 		 		 			4.95 ul 		 		 			8.25 ul 		 		 			13.2 ul 		 	  		 			total 		 		 			51.5 			ul 		 		 			  			 		 		 			165 ul 		 		 			275 ul 		 		 			440 ul 		 	 
 

SECTION: Step 2
43. Add to the QC tube 51.5 μL from the master mix of the digest buffer
SECTION: Step 2
44. Pipette up and down to fully mix. 

Incubate the QC tube in an agitating thermal mixer set at 1,250 rpm as follows: 

SECTION: Step 2
44.1.  
      								 									Temp 								 								 									Time 								 							  								 									55 								 								 									15 minutes 								 							  								 									68 								 								 									45 minutes 								 							  								 									25 								 								 									hold 								 							 
 
SECTION: Step 2
45. (make sure the samples go down to 25° as well)
SECTION: Step 2
46. Pipette up and down to fully mix. 

Incubate the QC tube in an agitating thermal mixer set at 1,250 rpm as follows:
SECTION: Step 2
47. Purify the QC sample using Zymo Research DNA Clean and ConcentratorTM-5 Kit (DCC). 

Quick spin your QC tube, add 200 μL of DCC DNA Binding Buffer, and mix thoroughly.

  

SECTION: Step 2
48. Transfer the mixture to the Zymo-SpinTM Column placed in a collection tube.
SECTION: Step 2
49. Centrifuge for 30 seconds at 13,000 x g. 

Discard the flow-through.
SECTION: Step 2
50. Add to the column 200 μL DCC DNA Wash Buffer 

SECTION: Step 2
51. Centrifuge for 1 minute at 13,000 x g.

 Discard the flow-through.
SECTION: Step 2
52. Repeat steps 7 and 8 once, for a total of 2 washes.
SECTION: Step 2
53. Transfer the column to a new 1.5 mL tube.
SECTION: Step 2
54. Add 10 μL DCC DNA Elution Buffer directly to the column and incubate for 1 minute at room temperature.
SECTION: Step 2
55. Centrifuge for 1 minute at 13,000 x g. Discard the column. 

Your 1.5 mL tube now contains your purified QC DNA.
SECTION: Step 2
56. Quantify the purified QC DNA with a Qubit® Fluorometer and Qubit® dsDNA HS 	Kit. Based on the Qubit concentration, the total lysate amount (ng) can be calculated as follows: 

Total Lysate (ng) = Qubit reading ng/μL x 10 μL (elution volume) x 23.4 (dilution factor) 

You will use in Stage 3 a volume of the lysate that corresponds to 1,000 ng. This volume can be calculated as follows: 

Volume 	(μL) = 1000 (ng) x 58.5 (μL) / Total Lysate (ng) 

If the total lysate amount is < 1,000 ng, use all of the lysate in Stage 3. 
SECTION: Step 2
57. Check the fragment size distribution of your purified QC sample on a TapeStation D5000 HS ScreenTape. Make sure your sample is diluted to 1 ng/μL. 
For 	optimal nucleosome-level resolution, the digestion profile should contain 40% - 70% mononucleosomes: the first DNA peak, typically in the size range of 50 - 250 bp for TapeStation, should account for 40% - 70% of total DNA (Figure 3). The size range of the peak may vary for other analytical instruments such as Bioanalyzer and 	Fragment Analyzer. If the digestion profile contains 40% - 70% mononucleosomes, proceed to Stage 3: 	Proximity Ligation, page 16. 

If  the digestion profile contains < 40% mononucleosomes, do not 	proceed with the rest of the protocol. In this case, re-start the protocol and use 2 μL of  MNase Enzyme Mix instead of 0.5 μL in step 14 in Stage 1: Crosslinking, Digestion and Lysis, page 11. 	

If the digestion profile contains > 70%, you may proceed to Stage 3: Proximity Ligation, page 16 with 	caution. The library may include a reduced proportion of long-range information. This profile is likely due to suboptimal cell counting or a significant cell loss in the washing steps after crosslinking (steps 9 to 12 in Stage 1: Crosslinking, Digestion and Lysis, page 11). 	

SECTION: pre-step 3
58. Stage 3: Proximity Ligation 

As you prepare for Stage 3, keep the following in mind: 

Proximity ligation takes ~ 4.5 hours. 

Follow best practices when working with beads 
SECTION: Step 3
63. 3.1 Bind Chromatin to Chromatin Capture Beads 

Follow the steps below for Bind Chromatin to Chromatin Capture Beads: 
SECTION: Step 3
64. Equilibrate the Chromatin Capture Beads to room temperature and vortex thoroughly (>30 seconds) to resuspend.
SECTION: Step 3
65. Transfer 100 μL of resuspended Chromatin Capture Beads to a new 1.5 mL tube.
SECTION: Step 3
66. Add 1,000 ng of the lysate that was stored at -80° (step 1 NOTE in Stage 2: Lysate QC, page 14) to the 1.5 mL tube containing the beads. 

If the total lysate amount is <1,000 ng, add all of the lysate.
SECTION: Step 3
67. Pipette up and down to fully mix. Incubate at room temperature, off the magnetic rack, for 10 minutes.
SECTION: Step 3
68. Place the tube in the magnetic rack for 5 minutes (or until the solution looks clear). 

Discard the supernatant.
SECTION: Step 3
69. Remove the tube from the magnetic rack and wash the beads with 150 μL 1X Wash Buffer.

Pipette up and down to resuspend the beads, place the tube in the magnetic rack for 1 minute and discard the supernatant.
SECTION: Step 3
70. Repeat step 6 once, for a total of 2 washes.
SECTION: Step 3
71. 3.2 End polishing
Follow the steps below for End Polishing: 

Remove the tube from the magnetic rack and add to the beads 53.5 μL of a 	master mix containing the following reagents:

SECTION: Step 3
71.1.  
              		 			Reagents 		 		 			1X 		 		 			10% 			extra 		 		 			3X 		 		 			5X 		 		 			8X 		 	  		 			End 			Polish Buffer 		 		 			50 			ul 		 		 			55 			ul 		 		 			165 			ul 		 		 			275 			ul 		 		 			440 			ul 		 	  		 			End 			Polishing Enzyme Mix 		 		 			3.5 			ul 		 		 			3.85 			ul 		 		 			11.55 			ul 		 		 			19.25 			ul 		 		 			30.8 			ul 		 	  		 			Total 		 		 			53.5 			ul 		 		 			  			 		 		 			  			 		 		 			  			 		 		 			  			 		 	 
 
SECTION: Step 3
72. Pipette  up and down to fully mix. 

Incubate in an agitating thermal mixer set at 1,250 rpm as follows: 

SECTION: Step 3
72.1.  
      								 									Temp 								 								 									Time 								 							  								 									22 								 								 									30 									minutes 								 							  								 									65 								 								 									30 									minutes 								 							 
 
SECTION: Step 3
73. Allow the tube to reach room temperature then place it in the magnetic rack for 1 minute (or until the solution looks clear). 

Discard the supernatant. 

SECTION: Step 3
74. Remove the tube from the magnetic rack and wash the beads once with 150 μL 1X Wash Buffer. 

Pipette up and down to resuspend the beads, place the tube in the magnetic rack. 

Do not remove and discard the supernatant at this step. 

Keep the tube in the magnetic rack and the beads in buffer to ensure they do not dry out while you prepare for the next reaction. 

SECTION: Step 3
75. 3.3 Bridge Ligation 

Follow the steps below for Bridge Ligation: 

Prepare and place on ice fresh 50 μL Bridge Ligation Mix by mixing the 	following reagents: 

SECTION: Step 3
75.1.  
              		 			Reagents 		 		 			1X 		 		 			10% 			extra 		 		 			3X 		 		 			5X 		 		 			8X 		 	  		 			UltraPure 			water 		 		 			35 			ul 		 		 			38.5 			ul 		 		 			115,5 			ul 		 		 			192,5 			ul 		 		 			308 			ul 		 	  		 			5X 			bridge ligation buffer 		 		 			10 			ul 		 		 			11 			ul 		 		 			33 			ul 		 		 			55 			ul 		 		 			88 			ul 		 	  		 			Bridge 		 		 			5 			ul 		 		 			5.5ul 		 		 			16.5 			ul 		 		 			27.5 			ul 		 		 			44 			ul 		 	  		 			Total 		 		 			53.5 			ul 		 		 			  			 		 		 			  			 		 		 			  			 		 		 			  			 		 	 
 
SECTION: Step 3
76. Remove and discard the supernatant from step 4 in 3.2 End Polishing, page 17. 

Remove the tube from the magnetic rack and add to the beads: 

SECTION: Step 3
76.1.  
      								 									Reagent 								 								 									Volume/reaction 								 							  								 									Bridge 									Ligation mix 								 								 									50 									ul 								 							  								 									Bridge 									ligase 								 								 									1 									ul 								 							  								 									Total 								 								 									51 									ul 								 							 
 
SECTION: Step 3
77. Pipette up and down to fully mix.

 Incubate at 22° for 30 minutes in an agitating thermal mixer set at 1,250 rpm.
SECTION: Step 3
78. Place the tube in the magnetic rack for 1 minute (or until the solution looks clear). 

Discard the supernatant. 

SECTION: Step 3
79. Remove the tube from the magnetic rack and wash the beads once with 150 μL 1X Wash Buffer. 

Pipette up and down to resuspend the beads, place the tube in the magnetic rack for 1 minute and discard the supernatant. 

SECTION: Step 3
80. 3.4 Intra-Aggregate Ligation 

Follow the steps below for Intra-Aggregate Ligation: 

Remove the tube from the magnetic rack and add to the beads 52 μL of a master mix containing the following reagents: 

SECTION: Step 3
80.1.  
              		 			Reagents 		 		 			1X 		 		 			10% 			extra 		 		 			3X 		 		 			5X 		 		 			8X 		 	  		 			Intra-Aggregate 			Ligation Buffer 		 		 			50 			ul 		 		 			55 			ul 		 		 			165 			ul 		 		 			275 			ul 		 		 			440 			ul 		 	  		 			Intra-Aggregate 			Ligation enzyme mix 		 		 			2 			ul 		 		 			2.2 			ul 		 		 			6.6 			ul 		 		 			11 			ul 		 		 			17.6 			ul 		 	  		 			Total 		 		 			53.5 			ul 		 		 			  			 		 		 			  			 		 		 			  			 		 		 			  			 		 	 
 
SECTION: Step 3
81. Pipette up and down to fully mix. Incubate at 22° for 1 hour in an agitating thermal mixer set at 1,250 rpm. 


SAFESTOP For convenience, this ligation reaction can proceed overnight at 22° in an agitating thermal mixer set at 1,250 rpm. 

SECTION: Step 3
82. Place the tube in the magnetic rack for 1 minute (or until the solution looks clear). 

Discard the supernatant. 

SECTION: Step 3
83. 3.5 Crosslink Reversal 

Follow the steps below for Crosslink Reversal: 

Remove the tube from the magnetic rack and add to the beads 51.5 μL of a 	master mix containing the following reagents (in order): 

SECTION: Step 3
83.1.  
              		 			Reagents 		 		 			1X 		 		 			10% 			extra 		 		 			3X 		 		 			5X 		 		 			8X 		 	  		 			UltraPure 			water 		 		 			45 			ul 		 		 			49.5 			ul 		 		 			148.5 			ul 		 		 			247.5 			ul 		 		 			396 			ul 		 	  		 			10% 			crosslink reversal buffer 		 		 			5 			ul 		 		 			5.5 			ul 		 		 			165 			ul 		 		 			275 			ul 		 		 			44 			ul 		 	  		 			Proteinase 			K 		 		 			1.5 			ul 		 		 			1.65 			ul 		 		 			4.95 			ul 		 		 			8.25 			ul 		 		 			13.2 			ul 		 	  		 			Total 		 		 			51.5 			ul 		 		 			  			 		 		 			  			 		 		 			  			 		 		 			  			 		 	 
 
SECTION: Step 3
84. Pipette up and down to fully mix. Incubate in an agitating thermal mixer set at 1,250 rpm as follows: 

SECTION: Step 3
84.1.  
      							 								Temp 							 							 								Time 							 						  							 								55 							 							 								15 								minutes 							 						  							 								68 							 							 								45 								minutes 							 						  							 								25 							 							 								hold 							 						 
 
SECTION: Step 3
84.2. SAFESTOP; For convenience, you can hold at 25° overnight in an agitating thermal mixer set at 1,250 rpm.
SECTION: Step 3
85. Place the tube in the magnetic rack for 1 minute. Transfer 50 μL of the SUPERNATANT to a new 1.5 mL tube. 

Discard the beads. 

SECTION: Step 3
86. 3.6 DNA Purification
Follow the steps below for DNA Purification on SPRIselect Beads:


Vortex the SPRIselect beads thoroughly (>30 seconds) to resuspend.

  

SECTION: Step 3
87. Add 90 μL of resuspended SPRIselect beads to the 1.5 mL tube containing your sample.
SECTION: Step 3
88. Vortex to mix thoroughly. 

Quick spin the tube (make sure to spin down only briefly and to stop before the beads start to settle).
SECTION: Step 3
89. Incubate the tube at room temperature, off the magnetic rack, for 5 minutes.
SECTION: Step 3
90. Quick spin the tube and place it in the magnetic rack for 5 minutes. Discard the supernatant.


SECTION: Step 3
91. Leave the tube in the magnetic rack and wash the beads twice with 200 μL fresh 80% ethanol.

 Do not resuspend the beads for these washes. 

Simply add the ethanol, wait for 1 minute then discard the ethanol supernatant.
SECTION: Step 3
92. After the last wash, quick spin the tube and place it in the magnetic rack for 1 minute. 

Use a 10 μL pipette tip to remove traces of ethanol
SECTION: Step 3
93. Air dry the beads for 5 minutes in the magnetic rack until no residual ethanol remains. 

Do not over dry the beads.
SECTION: Step 3
94. Off the magnetic rack, resuspend the beads in 52 μL TE Buffer pH 8.0.
SECTION: Step 3
95. Vortex to mix thoroughly. 

Quick spin the tube (make sure to spin down only briefly and to stop before the beads start to settle).
SECTION: Step 3
96. Incubate at room temperature, off the magnetic rack, for 5 minutes.
SECTION: Step 3
97. Quick spin the tube and place it in the magnetic rack for 1 minute.
SECTION: Step 3
98. Transfer 50 μL of the SUPERNATANT (purified DNA) to a new tube. Discard the beads.
SECTION: Step 3
99. Quantify the purified DNA using a Qubit Fluorometer and Qubit dsDNA HS Kit. 

You should recover a minimum of 50 ng to proceed to Step 4: Library Preparation
SECTION: Step 3
100. You will use 150 ng of your purified DNA for Stage 4 in a 50 μL volume. If needed, you can bring up the volume to 50 μL using TE Buffer pH 8.0.
	If you recovered <150 ng, use all of the purified DNA to proceed to Stage 4.
	If you recovered >150 ng, use 150 ng to proceed to Stage 4 and keep the remaining purified DNA stored at -20° You can use the remaining




SAFESTOP ; Purified DNA sample can be stored at -20° for up to 6 months.
SECTION: pre-step 4
101. Stage 4: Library Preparation
As you prepare for Stage 4, keep the following in mind:

The library preparation protocol does not require fragmentation.

The library preparation protocol takes ~ 2.5 hours.

  

SECTION: Step 4
105. 4.1 End Repair


Follow the steps below for End Repair:


Place 50 μL of purified DNA (150 ng) in a 0.2 mL PCR tube.
SECTION: Step 4
106. Add to the PCR tube 10.5 μL of a master mix containing the following reagents:
SECTION: Step 4
106.1.  
              		 			Reagents 		 		 			1X 		 		 			10% 			extra 		 		 			3X 		 		 			5X 		 		 			8X 		 	  		 			End 			repair buffer 		 		 			7 			ul 		 		 			7.7 			ul 		 		 			23.1 			ul 		 		 			38.5 			ul 		 		 			61.6 			ul 		 	  		 			End 			repair enzyme mix 		 		 			3 			ul 		 		 			3.3 			ul 		 		 			9.9 			ul 		 		 			16.5 			ul 		 		 			26.4 			ul 		 	  		 			250 			mM DTT 		 		 			0.5 			ul 		 		 			0.55 			ul 		 		 			1.65 			ul 		 		 			2.75 			ul 		 		 			4.4 			ul 		 	  		 			Total 		 		 			10.5 			ul 		 		 			  			 		 		 			  			 		 		 			  			 		 		 			  			 		 	 
 
SECTION: Step 4
107. Pipette up and down to fully mix. 

Quick spin the tube.
SECTION: Step 4
108. Incubate in a thermal cycler as follows:
SECTION: Step 4
108.1.  
      							 								Temp 							 							 								Time 							 						  							 								20 							 							 								30 minutes 							 						  							 								65 							 							 								30 minutes 							 						  							 								12 							 							 								hold 							 						 
 
SECTION: Step 4
109. 4.2 Adapter Ligation and USER Digest


Follow the steps below for Adapter Ligation and USER Digest:


Add to the PCR tube containing the end-repaired sample the following reagents:
SECTION: Step 4
109.1.  
      								 									Reagent 								 								 									Volume/reaction 								 							  								 									Adaptor 									for Illumina 								 								 									2.5 									ul 								 							  								 									Ligation 									Enzyme mix 								 								 									30 									ul 								 							  								 									Ligation 									Enhancer 								 								 									1 									ul 								 							  								 									Total 								 								 									33.5 									ul 								 							 
 
SECTION: Step 4
110. Pipette up and down to fully mix. 
Quick spin the tube.
SECTION: Step 4
109.2. NOTE The Ligation Enzyme Mix and Ligation Enhancer can be mixed ahead of time.  

The master mix is stable for 8 hours at 4°

We do not recommend adding the Adaptor for Illumina to the master mix.
SECTION: Step 4
111. Incubate at 20° for 15 minutes in a thermal cycler. 

Hold at 12°
SECTION: Step 4
112. Following incubation, add 3 μL of USER Enzyme Mix to the PCR tube.
SECTION: Step 4
113. Pipette up and down to fully mix. 

Quick spin the tube.
SECTION: Step 4
114. Incubate at 37 ° for 15 minutes in a thermal cycler. 

Hold at 12 °
SECTION: Step 4
115. 4.3 DNA Purification


Follow the steps below for DNA Purification:

Vortex the speed beads thoroughly (>30 seconds) to resuspend.
SECTION: Step 4
116. Add 80 μL of resuspended speed beads to the PCR tube containing the adaptor-ligated sample.
SECTION: Step 4
117. Vortex to mix thoroughly. 

Quick spin the tube (make sure to spin down only briefly and to stop before the beads start to settle).
SECTION: Step 4
118. Incubate the tube at room temperature, off the magnetic rack, for 5 minutes.
SECTION: Step 4
119. Quick spin the tube and place it in the magnetic rack for 5 minutes. Discard the supernatant.
SECTION: Step 4
120. Leave the tube in the magnetic rack and wash the beads twice with 200 μL fresh 80% ethanol. 

Do not resuspend the beads for these washes. 

Simply add the ethanol, wait for 1 minute then discard the ethanol supernatant.
SECTION: Step 4
121. Quick spin the tube and place it in the magnetic rack for 1 minute. Use a 10 μL pipette tip to remove traces of ethanol
SECTION: Step 4
122. Air dry the beads for 5 minutes in the magnetic rack until no residual ethanol remains. Do not over dry the beads.
SECTION: Step 4
123. Off the magnetic rack, resuspend the beads in 100 μL TE Buffer pH 8.0.
SECTION: Step 4
124. Vortex to mix thoroughly. 

Quick spin the tube (make sure to spin down only briefly and to stop before the beads start to settle).
SECTION: Step 4
125. Incubate at room temperature, off the magnetic rack, for 5 minutes.
SECTION: Step 4
126. Quick spin the tube and place it in the magnetic rack for 1 minute.
SECTION: Step 4
127. Transfer 95 μL of the SUPERNATANT (purified adaptor-ligated DNA) to a new tube. Discard the beads.


	SAFESTOP Purified DNA sample can be stored at -20° overnight.
SECTION: Pre-Step 5
128. Stage 5: Ligation Capture and Amplification


As you prepare for Stage 5, keep the following in mind:
The Ligation Capture and Amplification protocol takes ~ 2 hours.

  

SECTION: Step 5
131. 5.1 Streptavidin Beads Preparation
	NOTE This step does not involve any DNA sample.


Follow the steps below for Ligation Capture and Amplification:


Vortex the Streptavidin Beads vial thoroughly (> 30 seconds) to resuspend the beads. 

Transfer 25 μL of resuspended Streptavidin beads to a new 1.5 mL tube.
SECTION: Step 5
132. Place the 1.5 mL tube containing the beads in the magnetic rack for 5 minutes. 

Discard the supernatant.
SECTION: Step 5
133. Remove the tube from the magnetic rack and wash the beads with 200 μL TWB: pipette up and down to resuspend the beads and place the tube in the magnetic rack for 1 minute. 

Discard the supernatant.
SECTION: Step 5
134. Repeat step 3 once, for a total of 2 washes.
SECTION: Step 5
135. After the second wash, resuspend the beads in 100 μL 2X NTB. 

Pipette up and down to fully mix.
SECTION: Step 5
136. 5.2 Ligation Capture
Follow the steps below for Ligation Capture:

Transfer the 95 μL of purified adaptor-ligated DNA (from step 13 in 4.3 DNA Purification, page 24) to the 1.5 mL tube containing the Streptavidin beads resuspended in 100 μL of 2X NTB.
SECTION: Step 5
137. Vortex to mix thoroughly. Quick spin the tube (make sure to spin down only briefly and to stop before the beads start to settle).
SECTION: Step 5
138. Incubate at 25 ̊ for 30 minutes in an agitating thermal mixer set at 1,250 rpm.
SECTION: Step 5
139. 5.3 Wash Sample on Streptavidin Beads
	
NOTE For each of the washes below, remove the tube from the magnetic rack, add the indicated buffer to the beads, pipette up and down to resuspend the beads, place the tube in the magnetic rack for 1 minute, and discard the supernatant. Remove all of the supernatant between each wash; residual supernatant can interfere with the downstream PCR.


Follow the steps below for Wash Sample on Streptavidin Beads:


Quick spin the tube and place it in the magnetic rack for 1 minute. Discard the supernatant.
SECTION: Step 5
140. Wash the beads once with 200 μL LWB.
SECTION: Step 5
141. Wash the beads twice with 200 μL NWB.
SECTION: Step 5
142. Wash the beads twice with 200 μL 1X Wash Buffer.
SECTION: Step 5
143. 5.4 Index PCR

NOTE Not all PCR enzymes and master mixes are compatible for amplification in the presence of Streptavidin beads. Please use the HotStart PCR Ready Mix supplied in your Dovetail Kit (Box 2).


Follow the steps below for Index PCR:


After the last wash, remove the tube from the magnetic rack and add to the beads 45 μL of a master mix containing the following reagents:
SECTION: Step 5
144. Add 5 μL Index Primer to the PCR reaction. 

Use one Index Primer per PCR reaction 
SECTION: Step 5
143.1.  
              		 			Reagents 		 		 			1X 		 		 			10% 			extra 		 		 			3X 		 		 			5X 		 		 			8X 		 	  		 			UltraPure 			water 		 		 			15 			ul 		 		 			16.5 			ul 		 		 			45 			ul 		 		 			75 			ul 		 		 			120 			ul 		 	  		 			HotStart 			PCR Ready Mix 		 		 			25 			ul 		 		 			27.5 			ul 		 		 			75 			ul 		 		 			125 			ul 		 		 			200 			ul 		 	  		 			Universal 			PCR primer 		 		 			5 			ul 		 		 			5.5 			ul 		 		 			15 			ul 		 		 			25 			ul 		 		 			40 			ul 		 	  		 			Total 		 		 			45 			ul 		 		 			  			 		 		 			  			 		 		 			  			 		 		 			  			 		 	 
 
SECTION: Step 5
145. Pipette up and down to fully mix then transfer to a new 0.2 mL PCR tube.
SECTION: Step 5
146. Quick spin the tube (make sure to spin down only briefly and to stop before the beads start to settle).

 Place the tube into the thermal cycler and run the following program:
SECTION: Step 5
146.1.  
          					 						Step 					 					 						Temp 					 					 						Time 					 					 						cycles 					 				  					 						enzyme activation 					 					 						98 					 					 						3 minutes 					 					 						1 					 				  					 						denature 					 					 						98 					 					 						20 seconds 					 					 						12 					 				  					 						anneal 					 					 						65 					 					 						30 seconds 					 				  					 						Extend 					 					 						72 					 					 						30 seconds 					 				  					 						Extend 					 					 						72 					 					 						1 minute 					 					 						1 					 				  					 						  						 					 					 						12 					 					 						hold 					 					 						1 					 				 
 
SECTION: Step 5
147. 5.5 Size Selection
  
Follow the steps below for Size Selection:


Quick spin the PCR tube and place it in the magnetic rack for 1 minute.
SECTION: Step 5
148. Transfer 47 μL of the SUPERNATANT to a new 1.5 mL tube. 

Discard the beads.
SECTION: Step 5
149. Add 53 μL of TE Buffer pH 8.0 to the 1.5 mL tube to bring the volume of the sample in the tube to 100 μL.
SECTION: Step 5
150. Vortex the speed beads thoroughly (>30 seconds) to resuspend.
SECTION: Step 5
151. Add 50 μL of resuspended speed beads to the 1.5 mL tube containing your sample.  

SECTION: Step 5
152. Vortex to mix thoroughly. Quick spin the tube (make sure to spin down only briefly and to stop before the beads start to settle).
SECTION: Step 5
153. Incubate the tube at room temperature, off the magnetic rack, for 10 minutes.
SECTION: Step 5
154. Quick spin the tube and place it in the magnetic rack for 5 minutes.
SECTION: Step 5
155. Transfer 145 μL of the SUPERNATANT to a new 1.5 mL tube. Discard the beads.
SECTION: Step 5
156. Add 30 μL of resuspended Speed beads to the 1.5 mL tube containing your sample.  

SECTION: Step 5
157. Vortex to mix thoroughly. 

Quick spin the tube (make sure to spin down only briefly and to stop before the beads start to settle).
SECTION: Step 5
158. Incubate the tube at room temperature, off the magnetic rack, for 10 minutes.
SECTION: Step 5
159. Quick spin the tube and place it in the magnetic rack for 5 minutes. 

Discard the supernatant.
SECTION: Step 5
160. Leave the tube in the magnetic rack and wash the beads twice with 200 μL fresh 80% ethanol. 

Do not resuspend the beads for these washes. 

Simply add the ethanol, wait for 1 minute then discard the ethanol supernatant.
SECTION: Step 5
161. Quick spin the tube and place it in the magnetic rack for 1 minute. Use a 10 μL pipette tip to remove traces of ethanol.
SECTION: Step 5
162. Air dry the beads for 5 minutes in the magnetic rack until no residual ethanol remains. Do not over dry the beads.
SECTION: Step 5
163. Off the magnetic rack, resuspend the beads in 30 μL TE Buffer pH 8.0.
SECTION: Step 5
164. Vortex to mix thoroughly. 

Quick spin the tube (make sure to spin down only briefly and to stop before the beads start to settle).
SECTION: Step 5
165. Incubate the tube at room temperature, off the magnetic rack, for 5 minutes.
SECTION: Step 5
166. Quick spin the tube and place it in the magnetic rack for 1 minute.
SECTION: Step 5
167. Transfer 28 μL of the SUPERNATANT to a new 1.5 mL tube. The supernatant is your size selected library. Discard the beads.
SECTION: Step 5
168. Quantify your size selected library using a Qubit Fluorometer and Qubit dsDNA HS Kit.

SECTION: Step 5
169. Use a TapeStation or Bioanalyzer to verify the size distribution of your size selected library. 

The size range is expected to be between 350 bp and 1,000 bp.  

SAFESTOP The library can be stored at -20° for up to 6 months.
SECTION: Pre-Step 5
129. Before You Begin

Thaw Universal PCR Primer, Index Primer, and HotStart PCR Ready Mix at room
temperature. 

Vortex to mix prior to use.
SECTION: Pre-Step 5
130. Equilibrate TE Buffer pH 8.0, speed beads, Streptavidin Beads, TWB, 2X NTB, LWB, and NWB to room temperature.
SECTION: pre-step 4
102. Before You Begin

The End Repair Buffer may have precipitated in storage. 

Incubate for at least 10 minutes at 37° until there is no visible precipitate.
SECTION: pre-step 4
103. Equilibrate TE Buffer pH 8.0 and SPRIselect beads to room temperature.
SECTION: pre-step 4
104. Thaw 250 mM DTT and Adaptor for Illumina at room temperature. 

Vortex to mix prior to use.
SECTION: pre-step 3
60. Thaw End Polishing Buffer, 5X Bridge Ligation Buffer, Bridge, and Intra-Aggregate Ligation Buffer at room temperature.

Vortex to mix prior to use. 
SECTION: pre-step 3
59. Before You Begin 

10X Crosslink Reversal Buffer might have precipitated in storage. 

Incubate at 37° for 15 minutes or until the precipitate is no longer visible. Vortex to mix prior to use. 

SECTION: pre-step 3
61. Prepare fresh 80% ethanol for DNA purification with speed beads for optimal results. 

Fresh preparations of 80% ethanol will also be used in Stage 4, DNA Purification, page 24 and Stage 5, Size Selection, page 27. 

You need a minimum of 1 mL for all these stages. 
SECTION: pre-step 3
62. Equilibrate TE Buffer pH 8.0, Chromatin Capture Beads, and speed beads to room temperature.
SECTION: pre-step 2
37. Before You Begin 

Verify before use that 100% ethanol was added to the DNA Wash Buffer supplied in the Zymo Research DNA Clean & Concentrator-5 Kit, as directed by the manufacturer. 

SECTION: pre-step 2
38. Program the thermal mixer as follows:

SECTION: pre-step 2
38.1.  
      						 							Temp 						 						 							Time 						 					  						 							55 						 						 							15 							minutes 						 					  						 							68 						 						 							45 							minutes 						 					  						 							25 						 						 							Hold 						 					 
 
SECTION: pre-step 2
39. 10X Crosslink Reversal Buffer might have precipitated in storage. 

Incubate at 37° for 15 minutes or until the precipitate is no longer visible. 

Vortex to mix prior to use. 

SECTION: pre-Step 1
6. Before You Begin

The 10X Wash Buffer and 20% SDS might have precipitated in storage. 	

Incubate these solutions at 37° for 15 minutes or until the precipitate is no longer visible. 

Vortex to mix prior to use. 


SECTION: pre-Step 1
7. Dilute 10x wash buffer to 1x with ultrapure water. 

store at room temperature. 

1x wash buffer is stable at room temperature for 2 months. 

You need ~ 2 ml of 1x wash buffer per sample for the entire protocol.
SECTION: pre-Step 1
8. Prepare 3 M DSG in DMSO (anhydrous) by dissolving 1 mg of DSG in 10.22 μl DMSO. 

DSG is water-insoluble and moisture-sensitive. 

Prepare immediately before use. 

Do not store DSG in solution.

  
  

SECTION: pre-Step 1
9. Prepare fresh 1x nuclease digest buffer and store at room temperature. 

1x nuclease digest buffer is stable for 1 day at room temperature.

You need 50 μl of 1x nuclease digest buffer per sample. 

To prepare 1x nuclease digest buffer (50 μl), mix the following components:    
SECTION: pre-Step 1
10. Set 	the thermal mixer at 22°, shaking at 1,250 rpm. 
22 °C 
   

SECTION: pre-Step 1
11. Thaw 0.5 M EGTA at room temperature. 
  
Vortex to mix prior to use.
SECTION: pre-Step 1
9.1.  
              		 			Reagent 		 		 			1X 		 		 			10% 			extra 		 		 			3X 		 		 			5X 		 		 			8X 		 	  		 			UltraPure 			water 		 		 			40 ul 		 		 			44 ul 		 		 			132 ul 		 		 			220 ul 		 		 			352 ul 		 	  		 			10x 			Nuclease digest buffer 		 		 			5 ul 		 		 			5.5 ul 		 		 			16.5 ul 		 		 			27.5 ul 		 		 			44 ul 		 	  		 			100 mM 			MgCl2 		 		 			5 ul 		 		 			5.5 ul 		 		 			16.5 ul 		 		 			27.5 ul 		 		 			44 ul 		 	  		 			total 		 		 			50 ul 		 		 			  			 		 		 			165 ul 		 		 			275 ul 		 		 			440 ul 		 	 
 
SECTION: pre-experimentation
4. Ensure there is enough liquid nitrogen for the protocol