Covaris

truChIP® Chromatin Shearing Kit
Adaptive Focused Acoustics® (AFA®)-based
Chromatin Shearing for ChIP-based Applications
PN 520154 (with Formaldehyde) and PN 520127 (without Formaldehyde)

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Part Number: 010179 Rev N | November 2019

CovarisTable of Contents

Intended Use ........................................................................................................................................................................................ 3
Introduction .......................................................................................................................................................................................... 3
Revision History ................................................................................................................................................................................... 3
Procedure Overview ............................................................................................................................................................................ 4
Sample Input Requirements ............................................................................................................................................................... 4
Kit Contents .......................................................................................................................................................................................... 4
Storage ................................................................................................................................................................................................... 5
Supplied by User .................................................................................................................................................................................. 5
Protocol ................................................................................................................................................................................................. 6
A. Cell Preparation and Crosslinking ........................................................................................................................................... 6
A.1 Suspension Cells ................................................................................................................................................................ 6
A.2 Adherent Cells .................................................................................................................................................................... 8
B. Nucleic Preparation ................................................................................................................................................................... 10
C. Chromatin Shearing .................................................................................................................................................................. 11
D. Chromatin Shearing Efficiency Analysis ................................................................................................................................ 12
Supplemental Material ........................................................................................................................................................................ 13
Appendix A: AFA Focused-ultrasonicator Operating Conditions ........................................................................................... 13
Appendix B: Preparing Sheared Chromatin for IP ..................................................................................................................... 14
Appendix C: NEXSON8 .................................................................................................................................................................. 15
Appendix D: Additional Notes ...................................................................................................................................................... 15
References ............................................................................................................................................................................................. 16

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General Information
Intended Use

The truChIP Kit is intended for use in research applications (RUO). This product is not intended for the diagnosis, prevention, or
treatment of disease.

Introduction
The truChIP Chromatin Shearing Kit is optimized for the efficient and reproducible shearing of chromatin from adherent andsuspension mammalian cells using Covaris AFA Focused-ultrasonicators. Focused-ultrasonicators provide a non-contact and isothermal
method of shearing chromatin without compromising the structural integrity of the target epitopes of interest. This kit can be used toprepare samples for ChIP-qPCR, ChIP-ChiP, and ChIP-Seq applications.
truChIP is compatible with a variety of cell types including primary, stem cells, and sorted cells. The Covaris shearing buffer contains
SDS, which may not be compatible with immunoprecipitations (IP) using antibodies against some commonly used protein tags.
Therefore, Covaris recommends contacting the antibody manufacturer prior to using this kit. To start, Covaris recommends users to
perform a one-time fixation and shearing time course study to empirically determine the optimal treatment conditions.

Revision History
Part Number

Revision

Date

Description of Change

010179I

03/17

Update template and publish ME220 settings

010179J

7/17

Remove specific content description of Buffer D3010179K

7/17

Correct procedure overview

010179L

11/18

Correct step 10 on the nuclei prep protocol and add IP Dilution Buffer

010179M

6/19

Adding water level clarification

10/19

Change Protease inhibitor cocktail, change LE220 settings,
add Buffer for reverse X-link, change input cell numbers for
M-Series Instruments, adjust fixation times for TFs, added LE220-plus settings,
added IP part and description, added NEXSON settings as Appendix C

010179Patents Granted and Pending

N

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Procedure Overview
Collect cells and resuspend in fixing buffer

Crosslink Protein-DNA interactions with formaldehyde

Lyse cells and isolate nuclei

Wash nuclei and resuspend in shearing buffer

Wash nuclei and resuspend in shearing buffer

Lyse nuclei and shear chromatin with AFA Focused-ultrasonicator

Sample Input Requirements
The truChIP Chromatin Shearing Kit is compatible with a range of inputs from less than 1 million (1x10 6) and up to 200 million (2x108)
cells. The Low Cell protocol has been optimized for chromatin shearing of 1 to 3 million cells using the microTUBE-130 with AFA
Fiber; the High Cell protocol uses the milliTUBE–1 mL with AFA Fiber to process up to 30 million cells; and the batch protocol is
optimized for preparing up to 200 million cells cultured as a suspension for chromatin shearing in 6 separate milliTUBE–1 mL with AFA
Fiber.
Single Sample

Low CellHigh Cell

Batch

Input cell number

1 to 3 million* (1 to 3 x 106) cells

5 to 30 million* (0.5 to 3 x 107) cells

50 to 200 Million* (0.5 to 2 x 108) cells

Number of samples sheared per kit

50

15

2

AFA tubemicroTUBE–130

milliTUBE–1 mL with AFA Fiber

6 × milliTUBE–1 mL with AFA Fiber

Shearing volume

130 µl1 mL

6 x 1 mL

*Note: Please note that for M-Series Instruments we suggest the following upper limits for input cell numbers: Low Cell: 1.5M, High Cell: 15M, Batch: 90M

Kit Contents
Buffer A

7.5 mL

10X Fixing Buffer

Buffer B

5 mL

5X Lysis Buffer

Buffer C

2.5 mL

10X Wash Buffer

Buffer D3

6 mL

10X SDS Shearing Buffer

Buffer E

6 mL

1X Quenching Buffer

Buffer F

Protease Inhibitor Cocktail Tablet

50X Protease Inhibitor Cocktail (PIC) – See Note

Buffer G

1.0 mlReverse Crosslinking Buffer

Dilution Buffer

20 mL

2X IP Dilution Buffer

Formaldehyde

5 x 1 mL ampules (PN 520154 only)

16% Methanol-free Formaldehyde

Note: To prepare the 50X Protease Inhibitor Cocktail add 1 ml of 18 megohm (mΩ) water to the Protease Inhibitor Cocktail Tablet in vial and vortex the tube
until the powder is fully dissolved. The buffer is stable for 1 to 2 weeks at 2 to 8 °C and at least 12 weeks at -15 to 25 °C. We suggest to aliquot the buffer inworking aliquots for storage at -15 to 25 °C.
Safety Data Sheets: https://covaris.com/resources/safety-data-sheets/
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Storage

The kit is shipped at room temperature and should be stored at 2 to 8 °C upon arrival. Prior to use, kit reagent Buffers D3 and E may
have to be warmed to 55 °C to dissolve precipitate and then cooled to room temperature before use.
Note: Mix solutions well before use to ensure solutions are completely solubilized.

Supplied by User
• Molecular Biology Grade Water – Thermo Scientific (Cat. No. SH3053802), Mo Bio (Cat. No. 17012-200), or equivalent• Phosphate Buffered Salt Solution (PBS) – Mo Bio (Cat. No. 17330-500), Thermo Scientific (Cat. No. SH30256.FS), or equivalent• RNase A (DNase free) Thermo Scientific (Cat. No. EN0531) or equivalent• Proteinase K (RNase and DNase free) Thermo Scientific (Cat. No. 17916), NEB (Cat. No. P8102S), or equivalent• Covaris Focused-ultrasonicator (M220, ME220, S220, E220evolution, E220, or LE220-plus)• Refrigerated centrifuge having 15,000 x g capability• Rocker – Nutator® or equivalent• AFA Tubes, Holders, and Racks
Low Cell Protocol Consumables, Holder and Racks Required
Part Number

Description

M220 Holder &Insert

ME220 Holder &Insert

S-Series Holder

E220evolution
Rack

E220 Rack

LE220-plus Rack

520045

microTUBE AFA Fiber
Pre-Slit Snap-Cap

500414 & 500489

500514 & 500526

500114500433500111

N/A

520052

microTUBE AFA Fiber
Crimp-Cap

N/A

500514 & 500526

500114500433500282500282520216

microTUBE-130 AFA
Fiber Screw-Cap

500414 & 500489

500522 & 500534

500339

N/A

N/A

N/A

5200538 microTUBE Strip V1

N/A

500514 & 500526

N/A

500430500191500191520217

8 microTUBE-130 AFA
Fiber Strip V2

N/A

500518 & 500526

N/A

N/A

N/A

N/A

52007896 microTUBE Plate

N/A

N/A

N/A

N/A

No Rack Required

500329High Cell Protocol Consumables, Holder, and Racks Required
Part Number

Description

M220 Holder &Insert

ME220 Holder &Insert

S-Series Holder

E220evolution
Rack

E220 Rack

LE220-plus Rack

520130

milliTUBE–1 mL
with AFA Fiber

500414 & 500422

500520 & 500534

500371500431500368500368

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ProtocolA. Cell Preparation and Crosslinking

The truChIP protocol uses a two-step lysis method to ensure reproducible and efficient shearing of both suspension and adherent cells.
Follow the Cell Preparation and Crosslinking method (A.1 – Suspension and A.2 – Adherent) for your cell culture type.
Note: ChIP assays are sensitive to crosslinking and shearing conditions. Therefore, Covaris recommends users to include multiple fixation and shearing time points
to empirically determine the optimal treatment conditions.

A.1 Suspension Cells
1.		 Prepare solutions for the appropriate number of samples being processed before starting. Volumes in the table below are for			 a single sample.
BufferLow CellHigh Cell

Batch

Cold 1X PBS

Final Volume: 2.0 mL per sample- Store on ice

Final Volume: 4 mL per sample- Store on ice

Final Volume: 40 mL per batch- Store on ice

1X Fixing Buffer A

Final Volume: 0.5 mL per sample- Mix 50 µl of Fixing Buffer A with 0.450
mL of molecular biology grade water

Final Volume: 2 mL per sample- Mix 200 µl of Fixing Buffer A with 1.8
mL of molecular biology grade water

Final Volume: 20 mL per batch- Mix 2.0 mL of Fixing Buffer A with 18
mL of molecular biology grade water

Fresh 11.1% Formaldehyde

Final Volume: 1 mL per 1 to 20 samples- Mix 690 µl of 16% Fresh Formaldehydewith 310 µl of molecular biology grade
water

Final Volume: 1 mL per 1 to 5 samples- Mix 690 µl of 16% Fresh Formaldehydewith 310 µl of molecular biology grade
water

Final Volume: 2 mL per batch- Mix 1.38 mL of 16% Fresh
Formaldehyde with 0.62 mL of
molecular biology grade water

Quenching Buffer E

Place in a 55 °C water bath to dissolve crystals, then place at ambient

Important Notes• The use of fresh methanol-free formaldehyde is required to achieve reproducible results• The methanol-free formaldehyde ampule is for one-time use only–storage for later use is not recommended

2.		 Collect cells by centrifugation at 200 x g for 5 minutes at room temperature. Remove media and wash cells once with cold 1X
			 PBS and collect cells again by centrifugation.
			Note: Some cells do not pellet well at 200 x g. If a “spongy” pellet is not visible, increase speed at 100 x g intervals until a pellet is visible.
Reagent

Low CellHigh Cell

Batch

Cold 1X PBS

400 µl

1.5 mL

20 mL

Input cell number

1 to 3 x 106 Cells

1 to 3 x 107 Cells

0.5 to 2 x 108 Cells

Centrifuge Tube

2.0 mL

2.0 mL

50 mL

3.		 Re-suspend cells in room temperature Fixing Buffer A.
Reagent

Low CellHigh Cell

Batch

Fixing Buffer A

400 µl

1.5 mL

20 mL

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4.		 Crosslink cells by adding freshly prepared 11.1% formaldehyde solution to a final concentration of 1% and set timer.
			Note: The use of fresh methanol-free formaldehyde is required to achieve reproducible results.
Reagent

Low CellHigh Cell

Batch

Fresh 11.1% Formaldehyde

40 µl

150 µl2.0 mL5.		 Place cells on a shaking platform at room temperature for the recommended time.
			Note: We recommend including two fixation times. Typically, 2.5 and 5 minutes for stem, blood-derived and primary cells, and 5 and 10 minutes for all					other cell types. The fixation time will also depend on the respective target for IP and must be determined empirically. For transcription factors

					

a fixation time of 10 minutes is recommended.

6.		 Quench the crosslinking reaction by adding the appropriate volume of Quenching Buffer E to the fixed cells. Keep cells on
			 rocker at room temperature for an additional 5 minutes.
Reagent

Low CellHigh Cell

Batch

Quenching Buffer E

23 µl

87 µl

1.2 mL7.		 Collect cells by centrifuging at 500 x g for 5 minutes at room temperature.
8.		 Aspirate the supernatant and wash twice with cold 1X PBS.
Reagent

Low CellHigh Cell

Batch

Cold 1X PBS

300 µl

1.0 mL

5.0 mL9.		 Collect cells by centrifugation at 200 x g for 5 minutes, 4 °C.
10. Proceed to nuclei preparation and chromatin shearing steps.
			Note: You may flash-freeze the fixed cells in liquid nitrogen at this point and store at -80 °C for short periods of time (e.g., 2 to 3 days). Longer-term					storage is not recommended.

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A.2 Adherent Cells
1.		 Grow the proper number of cells to conduct a single ChIP assay or the initial time course until they are 80 to 90% confluent.
			Note: Cell densities provided above are estimates provided as a general guideline. Accurate cell densities for your cell lines should be determined .
35 mm Plate

60 mm Plate

100 mm Plate

150 mm Plate

Cell Density~0.8 x 106~2.0 x 106~5.5 x 106~15 x 106

ProtocolLow Cell

Low CellHigh Cell

High Cell

Number of Plates

1 to 3

1 to 2

2 to 5

1 to 22.		 Prepare solutions for the appropriate number of samples being processed fresh before starting. Volumes in the table below
			 are for a single sample.
Buffer

35 and 60 mm Dish

100 and 150 mm Plate

Cold 1X PBS

Final Volume: 14 mL per Plate- Store on ice

Final Volume: 25 mL per Plate- Store on ice

1X Fixing Buffer A

Final Volume: 2 mL per Plate- Mix 200 µl of Fixing Buffer A with 1.8 mL of molecularbiology grade water

Final Volume: 2 mL per sample- Mix 500 µl of Fixing Buffer A with 4.5 mL of molecularbiology grade water

Fresh 11.1% Formaldehyde

Final Volume: 1 mL per 1 to 20 samples- Mix 208 µl of 16% Fresh Formaldehyde with 92 µl of
molecular biology grade water

Final Volume: 1 mL per 1 to 5 samples- Mix 0.69 mL of 16% Fresh Formaldehyde with 0.31 mL of
molecular biology grade water

Quenching Buffer E

Place in a 55 °C water bath to dissolve crystals, then place at ambient

Important Notes• The use of fresh methanol-free formaldehyde is required to achieve reproducible results• The methanol-free formaldehyde ampoule is for one-time use only–storage for later use is not recommended

3.		 Remove media and wash each plate one time with cold 1X PBS.
Reagent

35 and 60 mm Plate

100 and 150 mm Plate

Cold 1X PBS

2 mL

5 mL

4.		 Remove PBS and add room temperature Fixing Buffer A to each dish.
Reagent

35 and 60 mm Plate

100 and 150 mm Plate

Fixing Buffer A

2 mL

5 mL

5.		 Crosslink cells by adding freshly prepared 11.1% formaldehyde solution to a final concentration of 1% and start timing the			 crosslinking reaction.
			Note: The use of fresh methanol-free formaldehyde is required to achieve reproducible results.
Reagent

35 and 60 mm Plate

100 and 150 mm Plate

Fresh 11.1% Formaldehyde

200 µl500 µlPatents Granted and Pending

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6.		 Place cells on a shaking platform at room temperature for the recommended time.

			Note: We recommend including two fixation times. Typically, 2.5 and 5 minutes for stem, blood-derived and primary cells, and 5 and 10 minutes for all					other cell types. The fixation time will also depend on the respective target for IP and must be determined empirically. For transcription factors

					

a fixation time of 10 minutes is recommended.

7.		 Quench the crosslinking reaction by adding the appropriate volume of Quenching Buffer E to fixed cells. Keep on a shaking
			 platform at room temperature for an additional 5 minutes.
Reagent

35 and 60 mm Plate

100 and 150 mm Plate

Quenching Buffer E

120 µl300 µl8.		 Completely aspirate the solution from the plate.
9.		 Add cold 1X PBS to each dish and scrape cells from the plate into a proper vessel.
Reagent

35 and 60 mm Plate

100 and 150 mm Plate

Cold 1X PBS

450 µl5 mL

Centrifuge Tube

2.0 mL tube

15 mL conical

10. Wash the plate with an additional volume of cold 1X PBS to collect any remaining cells.
Reagent

35 and 60 mm Plate

100 and 150 mm Plate

Cold 1X PBS

450 µl5 mL

Centrifuge Tube

2.0 mL tube

15 mL conical

11. Collect cells at 200 x g for 5 minutes, 4 °C.
			Note: Some cells do not pellet well at 200 x g. If a “spongy” pellet is not visible, increase speed at 100 x g intervals until a pellet is visible.

12. Wash cells twice by resuspending in cold 1X PBS, and collecting by centrifugation at 200 x g, 4 °C.
Reagent

35 and 60 mm Plate

100 and 150 mm Plate

Cold 1X PBS

450 µl5 mL

13. Carefully and completely aspirate the supernatant from the tube(s), and place on ice. Proceed to nuclei preparation and			 chromatin shearing steps.
			Note: You may flash-freeze the fixed cells in liquid nitrogen at this point and store at -80 °C for short periods of time (e.g., 2 to 3 days). Longer-term					storage is not recommended.

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B. Nuclei Preparation

1.		 Prepare the proper number of suspension or adherent cells according to Step 1 on page 7. Place the required number of AFA
			 tubes on ice to pre-chill while preparing samples to shear.
			Note: Prepare the correct volume of fresh solutions for the nuclei preparation and chromatin shearing prior to beginning. Substituting any of the					reagents or changing any steps will adversely affect shearing efficiency and reproducibility.

			Note: Volumes in the table below are for a single sample.
Low CellHigh Cell

Batch

Number of fixed cells

1 to 3 x 106*

1 to 3 x 107*

0.5 to 2 x 108*

AFA TubemicroTUBE-130

milliTUBE-1 mL

6 × milliTUBE-1 mL

			*Note: Please note that for M-series Instruments we suggest the following upper limits for input cell numbers:					

Low Cell: 1.5M, High Cell: 15M, Batch: 90M

BufferLow CellHigh Cell

Batch

1X Lysis Buffer B

Final Volume: 0.5 mL per sample- Mix 100 µl 5X Lysis Buffer B with 390µl of molecular biology grade water

Final Volume: 1.0 mL per sample- Mix 200 µl 5X Lysis Buffer B with 0.79
mL of molecular biology grade water

Final Volume: 10 mL per sample- Mix 2 mL 5X Lysis Buffer B with 7.9 mLof molecular biology grade water- Add 10 µl of 50X Buffer F- Add 20 µl of 50X Buffer F- Add 200 µl of 50X Buffer F- Store on ice- Store on ice- Store on ice

Final Volume: 0.5 mL per sample- Mix 50 µl 10X Wash Buffer C with 440µl of molecular biology grade water

Final Volume: 1.0 mL per sample- Mix 100 µl 10X Wash Buffer C with
0.89 mL of molecular biology grade water

Final Volume: 10 mL per sample- Mix 1.0 mL 10X Wash Buffer C with 8.9
mL of molecular biology grade water- Add 10 µl of 50X Buffer F- Add 20 µl of 50X Buffer F- Add 200 µl of 50X Buffer F- Store on ice- Store on ice- Store on ice

Final Volume: 1 mL per sample- Mix 100 µl 10X Shearing Buffer D3 with
0.89 mL of molecular biology grade water

Final Volume: 3 mL per sample- Mix 300 µl 10X Shearing Buffer D3 with
2.67 mL of molecular biology grade water

Final Volume: 10 mL per sample- Mix 1.0 mL 10X Shearing Buffer D3 with
8.9 mL of molecular biology grade water- Add 20 µl of 50X Buffer F- Add 60 µl of 50 X Buffer F- Add 200 µl of 50 X Buffer F- Store on ice- Store on ice- Store on ice

1X Wash Buffer

1X Shearing Buffer D3(1mM EDTA, 10mM
Tris-HCl pH 7.6,
0.1% SDS)

2.		 Add Lysis Buffer B containing 1× protease inhibitors to cross-linked cells to lyse plasma membrane, gently resuspend by			 aspirating/dispensing 4 times. If cells were frozen after formaldehyde fixation, thaw cells on ice first.
Reagent

Low CellHigh Cell

Batch

Lysis Buffer B

300 µl

1 mL

10 mL

3.		 If processing using Low Cell or High Cell volumes, then transfer to 1.5 mL microcentrifuge tube. If processing for batch, then			 transfer to a 15 mL conical tube.

4.		 Incubate for 10 minutes on a rocker at 4 °C.
			Note: For very hard to lyse cells the nuclear extraction incubation step can be substituted with AFA lysis per the NEXSON protocol 8. The AFA					mediated lysis settings are provided in Appendix D.

5.		 Collect intact nuclei by centrifugation at 1,700 x g for 5 minutes, 4 °C. Decant the supernatant without disturbing the nuclei
			pellet.

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6.		 Gently resuspend pellet in Wash Buffer C containing protease inhibitor (Buffer F) and incubate on a rocker for 10 minutes, 4 °C.
			Note: The purpose of this wash is to significantly dilute the salts remaining from the Wash Buffer. Shearing in the presence of high salt concentrations
					

may result in the reversal of formaldehyde cross-links during the process.
Reagent

Low CellHigh Cell

Batch

Wash Buffer C

300 µl

1 mL

10 mL

7.		 Collect nuclei by centrifugation at 1,700 x g for 5 minutes, 4 °C. Carefully remove and discard the wash solution, taking care			 not to disturb the nuclei pellet.
8.		 Gently rinse the sides of the tube with Shearing Buffer D3 containing Protease inhibitor (Buffer F). Slowly dispense the buffer			 down the entire circumference of the upper-inside of the tube, taking care not to disturb the nuclei pellet.
Reagent

Low CellHigh Cell

Batch

Shearing Buffer D3

300 µl1 mL

1.5 mL9.		 Collect nuclei by centrifugation at 1,700 x g for 5 minutes, 4 °C. Decant the supernatant without disturbing the nuclei pellet.
10. Repeat steps 8 and 9 an additional time. Carefully remove and discard the supernatant, taking care not to disturb the nuclei
			pellet.
C. Chromatin Shearing
1.		 Resuspend nuclei pellet in the Shearing Buffer D3 and transfer to appropriate AFA Tube(s).

2.

Reagent

Low CellHigh Cell

Batch

Shearing Buffer D3

130 µl

1 mL

6 mL

AFA Tube

1 x microTUBE

1 x milliTUBE

6 x milliTUBE

Shear chromatin with an AFA Focused-ultrasonicator with appropriate rack or holder; settings are provided in Appendix A.

			Note: Optimization of shearing time should be conducted whenever experimental parameters (e.g., cell type, cell number, or sample volumes) are					 changed.

3.		 If processing samples for Low Cell Chromatin Shearing Optimization in microTUBEs, please aliquot 130 µl of the nuclei
			 preparation into 6 microTUBEs for carrying out the shearing time course of 2, 4, 6, 8, 10, and 12 minutes.
			 If processing samples for High Cell Chromatin Shearing Optimization, please aliquot 1 mL of the nuclei into one milliTUBE–1
			 mL with AFA Fiber for carrying out the shearing time course. of 2, 4, 8, 12, 15, and 20 minutes.

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4.		 If processing samples using the milliTUBE–1 mL with AFA Fiber, you may process all time points of the time course study in			 the same tube according to the figure below. After each time point interval, remove 35 µl of the sample and place in a pre			 chilled microcentrifuge tube labeled with the total processing time – store on ice. Replace the removed volume with 35 µl of			 Covaris Shearing Buffer D3 before running the next time point.

Programmed Interval Processing Time (min)

2

2

4

4

3

5

Total Processing Time (min)

2

4

8

12

15

20

5.		 Place the milliTUBE in the holder/rack and process on the ultrasonicator for the next programmed interval processing time.
			 25 µl will be used for DNA shearing size range analysis, and 10 µl will be used for epitope integrity analysis using western.
			 Replace the removed volume with 35 µl of Covaris Shearing Buffer D3 before running the next time point.
			Note: Carefully remove any traces of foam from the solution and the cap prior to placing sample back in the ultrasonicator for the next shearing time
					

6.

point when carrying out the time course experiment in the milliTUBE-1mL with AFA fiber.

After shearing, transfer samples into a pre-chilled microcentrifuge tube and place on ice until all tubes are processed. If batch

			 processing using a high-throughput ultrasonicator (e.g. E220), samples can be maintained in the instrument’s water bath at
			 4 °C before and after processing.
			Note: Do not proceed to IP before analyzing the sheared chromatin to make sure it is at the correct size and distribution for your downstream analysis.
			Note: To check the efficiency of your shearing, reserve 25 µl of the sheared chromatin.
			Note: Sheared chromatin can be stored at 4 °C for up to 2 days.
			Note: Freezing sheared chromatin is not recommended. Freeze/thaw cycles reduce IP efficiency and reproducibility.

D. Chromatin Shearing Efficiency Analysis
1.		 Take a 25 µl aliquot of the sheared sample and transfer to 0.6 mL microcentrifuge tube.
2.		 Add 3 µl of Buffer G.
3.		 Add 1 µl of RNase A (10 mg/mL) and incubate at 37 °C for 30 min.
4.		 Add 1 µl of Proteinase K (10 mg/mL) and reverse crosslink by heating at 65 °C overnight in a PCR cycler with a heated lid.
5.		 Purify DNA using either a commercial column-based kit (e.g., Qiagen QIAquick PCR Purification Kit, Cat. No. 28104)
6.		 Elute from column or resuspend pellet with 50 µl of elution buffer (10 mM Tris-HCl, pH 8.5).
7.		 If analyzing on an agarose gel, add 1 volume of loading dye to 5 volumes of purified DNA.
			Note: The use of loading dye without Bromophenol Blue is recommended. Bromophenol.

8.		 Load 300 to 600 ng of purified DNA per lane of a 1.2% agarose TAE gel, and run at 30 Volts for 2.5 to 4 hours for optimal
			 resolution.
			Note: We strongly advise the use of a Bioanalyzer DNA 12000 chip for chromatin shearing smear analysis.

9.		 Analyze 1 µl of purified DNA on an Agilent 2100 BioAnalyzer to provide an accurate representation of the shearing size range
			 and distribution.

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Supplemental Material
Appendix A. AFA Focused-ultrasonicator Operating Condition
Low Cell Chromatin Shearing Protocol
Instrument

M220

ME220

S220

E220evolution

E220

LE220/LE220-plus

200 to 700

200 to 700

200 to 700

200 to 700

200 to 700

200 to 700

PIP

75

75

105

105

105

300

Duty Factor (%)

5

5

2

2

2

15

CPB

200

1000

200

200

200

200

Treatment Time (minutes)

2 to 20

2 to 20

2 to 122 to 122 to 122 to 12

Setpoint Temperature (C)1

7

9

6

6

6

6

Min/Max Temperature (C)

4/10

6/12

3/9

3/9

3/9

3/9

Max Cell Number (Million)

3M

3M

3M

3M

3M

3M

AFA Intensifier Required2

N/A

N/A

Integrated

Yes

Yes

N/A

Water Level (run)3

Full

9

12

6

6

6

Sample Volume (µl)4

130

130

130

130

130

130

Target Size (bp)

Important Notes
1. If using the S220, E220evolution, E220, or LE220, set the temperature on the external chiller 3 °C below the setpoint temperature for the run. The min/max is set in
SonoLab. 2. If intensifier is required, please ensure PN 500141 is used. 3. Water level should always be 1 mm below the neck of the microTUBE–130 cap.
4. Always fill the microTUBE-130 with 130 µl of sample.

High Cell Chromatin Shearing Protocol
Instrument

M220

ME220

S220

E220evolution

E220

LE220/LE220-plus

200 to 700

200 to 700

200 to 700

200 to 700

200 to 700

200 to 700

PIP

75

75

140

140

140

420

Duty Factor (%)

10

15

5

5

5

30

CPB

200

1000

200

200

200

200

Treatment Time (minutes)

2 to 20

2 to 20

2 to 20

2 to 20

2 to 20

5 to 30

Setpoint Temperature (C)1

7

9

6

6

6

6

Min/Max Temperature (C)

4/10

6/12

3/9

3/9

3/9

3/9

Max Cell Number (Million)

30M

30M

30M

30M

30M

30M

AFA Intensifier Required

N/A

N/A

N/A

No

No

N/A

Water Level (run)2

Full

9

8

5

0

-4

Sample Volume (ml) 3

1

1

1

1

1

1

Target Size (bp)

Important Notes
1. If using the S220, E220evolution, E220, or LE220, set the temperature on the external chiller 3 °C below the setpoint temperature for the run. The min/max is set in
SonoLab. 2. Water level should always be 1 mm below the neck of the milliTUBE–1 mL with AFA Fiber cap. 3. Always fill the milliTUBE–1 mL with AFA Fiber with
1.0 mL of sample.

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Covaris

Appendix B. Preparing Sheared Chromatin for IP

1.		 Before proceeding to IP, dilute the chromatin 1:1 with the Covaris 2X IP Dilution Buffer, and vortex for 3 seconds.
2.		 Centrifuge sample after the addition of the IP Dilution buffer at 10,000 x g for 5 minutes at 4 °C to prepare the supernatant			 for IP.
Chromatin Sheared in D3 BufferHome Brew Methods

1:1 with 2x Covaris
Dilution Buffer

NaCl (150 mM final)
Triton (1% final)IP

Sheared chromatin must be diluted prior to IP.
This is required to equilibrate the sheared chromatin in a buffer that is compatible with antibody binding. Therefore, dilute thechromatin sheared in the Covaris chromatin shearing buffer D3 1:1 with the 2X Dilution Buffer. Alternatively, if the chromatin sample
should not be diluted further, add NaCl to a final concentration of 150 mM and Triton to a final concentration of 1%. If you do not
adjust these conditions prior to IP the enrichment of your target will not occur, or it will be suboptimal.

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Appendix C. NEXSON

Covaris8

The efficiency of nuclei isolation can be enhanced by supporting cellular lysis with AFA. This has especially proven useful for difficult
to lyse cells.
1.		 Transfer the fixed cells resuspended in 1ml of Buffer B into 1 ml milliTUBEs
2.		 Process the samples for AFA-mediated lysis according to the settings in the table below
AFA-mediated Cell Lysis Operating Conditions for up to 12 Million cells.
Up to 12 million cells in milliTUBE 1 ml AFA Fiber (PN 520130)
Instrument

M220

ME220

S220

E220evolution

E220

LE220/LE220-plus

PIP

75

75

75

75

75

N/A

Duty Factor (%)

5

5

2

2

2

N/A

CPB

200

1000

200

200

200

N/A

Treatment Time (minutes)

30 sec to 5 min

30 sec to 5 min

30 sec to 5 min

30 sec to 5 min

30 sec to 5 min

30 sec to 5 min

Setpoint Temperature (C)1

7

9

6

6

6

6

Min/Max Temperature (C)

4/10

6/12

3/9

3/9

3/9

3/9

Max Cell Number (Million)

12M

30M

30M

30M

30M

30M

AFA Intensifier Required

N/A

N/A

N/A

No

No

N/A

Water Level (run)2

Full

9

8

5

0

-4

Sample Volume (ml) 3

1

1

1

1

1

1

Important Notes
1. Treatment time must be determined for the cell type of interest and nuclei lysis should be determined by microscopy as described in the original publication 8.
2. If using the S220, E220evolution, E220, or LE220, set the temperature on the external chiller 3 °C below the setpoint temperature for the run. The min/max is set in
SonoLab.
3. Water level should always be 1 mm below the neck of the milliTUBE–1 mL with AFA Fiber cap.
4. Always fill the milliTUBE–1 mL with AFA Fiber with 1.0 mL of sample.

3.		 Continue with the nuclei preparation protocol beginning with step 5 (Section B, page 10).
Appendix D. Additional Notes
1.		 The treatment settings listed in this document are recommended guidelines. Actual results may vary depending on the cell			 type and mass.
2.		 The Covaris process uses high intensity focused ultrasonic (HIFU) energy and as such is influenced by objects in the acoustic
			 path from the transducer surface to the fluid sample. For example, particles and bubbles in the water bath may scatter the			 acoustic energy from the sample. Replace the bath water daily and ensure that appropriate time has been allowed for			 degassing and water bath temperature to stabilize prior to use of the instrument.
3.		 Bubbles in the sample fluid in the tube may diminish the acoustic dose effectiveness. Be sure to fill the tubes slowly with the			 recommended volumes and avoid the use of additional detergents that may induce foaming.

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Covaris

References

1.		 Lee T.I., Johnstone S.E., Young R.A., Chromatin immunoprecipitation and microarray-based analysis of protein location. Nature
			 Protocols (2006) 1:729-748.
2.		 Ralph M Bernstein, Ph.D. and Frederick C. Mills, Ph.D., Laboratory of Immunology, Division of Therapeutic Proteins, CDER,
			 FDA, NIH Campus, Bethesda, MD. We very much appreciate their contribution to the shearing buffer SDS concentration
			 titration experiment, formaldehyde fixation reduction time, and initial evaluation of our protocols and reagents.
3.		 Park P.J.; ChIP-seq: advantages and challenges of a maturing technology. Nature Reviews Genetics (2009) 10: 669-680.
4.		 Stewart D., Tomita A., Shi Y.B., Wong J., Chromatin immunoprecipitation for studying transcriptional regulation in Xenopus
			 oocytes and tadpoles. Methods Mol Biol (2006) 322:165-182.
5.		 Haring M, Offerman S, Danker T, Horst I, Peterhansel C and Stam M; Chromatin immunoprecipitation: optimization,
			 quantitative analysis and data normalization, Plant Methods (2007), 3:11.
6.		 Mukhopadhyay A, Deplancke B, Walhout AJM and Tissenbaum HA; Chromatin Immunoprecipitation (ChIP) coupled to			 detection by quantitative real-time PCR to study transcription factor binding to DNA in Caenorhabditis elegans. Nature
			 Protoc. (2008), 3(4) 698-70.
7.		 Das P.M, Ramachandran K., vanWert J, Signal R.; BioTechniques (2004), 37:961-969.
8.		 Arrigoni L, Richter A, Betancourt E, Bruder K, Diehl S, Manke T,1and Bönisch U; Standardizing chromatin research: a simple
			 and universal method for ChIP-seq. NAR. (2016), Vol. 44, No. 7

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