TITLE: CHIP-reCHIP Protocol for Mapping Bivalent Chromatin
AUTHORS: Janith A Seneviratne, William W H Ho, Eleanor Glancy, Melanie A Eckersley-Maslin

[DESCRIPTION]
This is a detailed protocol for performing sequential ChIP-reChIP to map H3K4me3-H3K27me3 bivalent chromatin regions. It has been optimised using mouse embryonic stem cells and so may need to be refined based on your cell type of interest. For more details please refer to the accompanying manuscript (Ho et al. 2023). 

[BEFORE_START]
Prepare Buffers as outlined below: 
NP buffer
Complete Chromatin Immunoprecipitation buffer
Low salt wash buffer
High salt wash buffer
LiCl wash buffer
Elution buffer

 NP BUFFER
 

CHROMATIN PRECIPITATION BUFFER
 
 
LOW SALT WASH BUFFER
 
 

HIGH SALT WASH BUFFER
 
 

LiCl WASH BUFFER
 
 

ELUTION BUFFER
 
 

[GUIDELINES]
Note: As a standard throughout the protocol, we recommend using low bind tubes and tips, and advise that all buffers be prepared using RNase and DNase free reagents. Furthermore, while most commercially available antibodies will have a recommended volume to use, we recommend validating and titrating antibody volumes and cell numbers prior to starting. Finally, we recommend designing and testing quantitative PCR primers to amplify positive and negative control regions, ensuring they have good efficiencies and melt-curves. PCR products should be between 80-120 base pairs. Validated primer sequences for mouse Embryonic Stem Cells can be found in the accompanying manuscript.


GENERAL WORKFLOW

A.    Chromatin fixation (2 hours)
B.    Chromatin fragmentation and Primary antibody incubation (2 hours hands on time with overnight incubation)
C.     First elution, buffer exchange and secondary antibody incubation (2 hours hands on time with overnight incubation)
D.    Secondary elution, de-crosslinking, and DNA purification (4 hours)

 KEY REAGENTS & RESOURCES
 
 


[STEPS]
SECTION: Key Resources &amp; reagents
1.   
  
  
  
  
   
  
  
  
  

 

 

 

SECTION: Chromatin Fixation
2. 
 

 

 



 

SECTION: Chromatin Fixation
3. Grow cells until they are 70-80% confluent across several plates. One will be used for counting and the remaining for fixation (collection plates).
SECTION: Chromatin Fixation
4. Collect and count cells on the counting plate: Remove media from the counting plate and wash once with DPBS.
SECTION: Chromatin Fixation
5. Add 1 mLof Trypsin or appropriate dissociation reagent per 10cm plate and incubate until cells lift off plate as single cells.

SECTION: Chromatin Fixation
6. Quench trypsin with 5 mL of media.

SECTION: Chromatin Fixation
7. Take aliquot and count cells to determine total cell number per plate and therefore the total number of cells overall.
SECTION: Chromatin Fixation
8. Prepare 1 %(w/v) formaldehyde solution by adding 0.625 mL of 16 %(w/v) formaldehyde solution to 9.375 mLof prewarmed DMEM per 10cm collection plate. 

 


SECTION: Chromatin Fixation
9. Remove media and wash cells on collection plates with DPBS.
SECTION: Chromatin Fixation
10. Cross link cells by adding 10 mL of 1 %(w/v) formaldehyde solution per plate of adherent cells for 8 min at room temperature.

SECTION: Chromatin Fixation
11. Quench with 1ml 1 Molarity (M) per plate of adherent cells to a final concentration 125 millimolar (mM), for 5 min at room temperature.

SECTION: Chromatin Fixation
12. Pour off medium and wash cells with 10 mLof ice cold DPBS.

SECTION: Chromatin Fixation
13. Pour off DPBS, scrape cells in residual PBS and transfer to low-bind 1.5ml tube. Pool cells from all plates into one tube.
SECTION: Chromatin Fixation
14. Centrifuge for at 500xg at 4 degrees for 10 minutes 500 x g, 10 min, 4 °C.

SECTION: Chromatin Fixation
15. Remove supernatant and resuspend cells to 2x10^7 cells per 1ml in PBS/5mM EDTA containing protease inhibitors. Aliquot by adding 0.1 mL cell slurry (corresponding to 2x10^6 cells) per low-bind tube.

SECTION: Chromatin Fixation
16. Pellet aliquots by centrifugation at 500xg at 4 degrees for 5 minutes 500 x g, 5 min, 4 °C.

SECTION: Chromatin Fixation
17. Remove supernatant and snap freeze on dry ice or in liquid nitrogen. Store pellets at -80 °C.

SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
18. 
 

 


 

 

SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
19. Bead preparation – 30 minutes
Prepare a sufficient amount of Protein A dynabeads for chromatin preclear and antibody binding. For each sample in the reChIP experiment you will need 150 µL. This corresponds to:

20 µL of beads to preclear 2x10^6 cells.
120 µL of beads for antibody-complex formation (20 µL per antibody-complex of which there are 6 reactions in total: 2xIgG, 2x H3K27me3, 2xH3K4me3)
10 µL for pipetting errors
SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
19.1. 
Take 150 mLProtein A dynabeads, place on magnetic rack and remove supernatant. 

SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
19.2. Remove tube from rack and resuspend in 500 µLof cold ChIP buffer containing freshly added protease inhibitor cocktail, discard buffer. 

     Repeat steps 2-3 for a total of 3 washes.
     After 3 washes resuspend in 150 µLof cold ChIP buffer.
     Keep chilled on ice until needed later.

SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
20. Binding Antibody to beads – 15 minutes preparation time plus at least 3 hours incubation
Label 6 low-bind tubes (2X IgG, 2X H3K4me3 and 2X H3K27me3)
SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
20.1. Add 500 µL of cold ChIP buffer to each tube.

SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
20.2. Add 20 µL of pre-washed Protein A dynabeads to each tube.

SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
20.3. Add appropriate antibody to each tube - 1 µg IgG (Invitrogen), 10 µLH3K27me3 (CST 9733), 2 µLH3K4me3 (Millipore 07-473). 

Note: If using other antibodies the volumes will need to be titrated to maximise signal:noise. Adding too little antibody will not capture all chromatin containing the modification of interest. Adding too much antibody increases the background non-specific binding.

SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
20.4. Incubate bead/antibody mix at 4 °C on a rotator for at least 3 hours 180 min.

SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
21. Chromatin preparation – 1 hour preparation time plus at least 3 hours incubation
SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
21.1. Thaw 1 vial of 2x10^6 crosslinked cells on ice
SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
21.2. Resuspend cell pellet (2x10^6 cells) in 97.68 µL NP buffer supplemented with 0.7 µLof 55 millimolar (mM) beta-mercaptoethanol, 1.82 µL of 0.1 Molarity (M) spermidine and freshly added protease inhibitor cocktail.

SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
21.3. Fragment chromatin with MNase: prepare MNase master mix (20 µL per sample)
12 µL 10X MNase buffer
1.76 µL 100mM DTT 
3.84 µL dH20
2.4 µL MNase




SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
21.4. Add 20 µL of MNase master mix to each tube of 2x10^6 cells in NP buffer.

SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
21.5. Incubate at 37 °C with shaking at   for 7.5 - 15 min.

 

SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
21.6. During digestion prepare STOP buffer: 
15 µL 100 millimolar (mM) EDTA
15 µL 1 % (v/v) / 1 % (v/v) solution
per sample

SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
21.7. Add 26.4 µL of STOP buffer to each sample to stop the MNase digestion.
SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
21.8. Incubate on ice for 5 minutes. 5 min 

SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
21.9. Vortex each tube for 30 seconds each.
SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
21.10. Bring volume up to 600 µL by adding 473.6 µL cold ChIP buffer containing protease inhibitor cocktail.

SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
21.11. Add 20 µL of pre-washed protein A Dynabeads from step 19 to the chromatin and incubate for at least 3 hours at 4 °C on rotator to pre-clear the chromatin. This is critical to reduce non-specific binding and decrease background signal. 180 min 

SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
22. Overnight incubation with primary antibody – 30 min and overnight
SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
22.1. Take all bead-antibody tubes from the 4-degree rotator.
SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
22.2. Back at the bench place the pre-cleared chromatin sample and 1x IgG, 1 x H3K4me3 and 1 x H3K27me3 bead-antibody complexes per sample on the magnet rack until the solution clears. 

 

SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
22.3. The remaining bead-antibody mixes can be stored at 4 degrees until needed on day 2.
SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
22.4. Take 10 µL of pre-cleared chromatin supernatant to a separate tube and label as 5% input control. Keep at 4 °C until day 3.

SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
22.5. Remove the supernatant from the antibody-bead complexes on the magnetic rack and discard.
SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
22.6. Add 200 µL of the pre-cleared chromatin supernatant to each antibody/bead mixture.

SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
22.7. Top up each chromatin/antibody/bead mixture with 300 µL of ChIP buffer to final volume of 500 µL. 

SECTION: Chromatin fragmentation and primary antibody incubation - Day 1
22.8. Incubate 15 min at 4 °C on a rotator. 

SECTION: First elution, buffer exchange and secondary antibody incubation – Day 2
23. 
 


Collect chromatin-antibody-bead samples from overnight 4 °C rotation.

SECTION: First elution, buffer exchange and secondary antibody incubation – Day 2
24. Wash chromatin-antibody-bead complexes a total of 9 times using the following steps keeping the samples cool by either placing magnet on ice or working in a cold room: 

a.     Place tubes on magnets rack and wait for solution to turn clear. 
b.     Carefully remove supernatant while on magnet making sure you do not disturb the beads. Make sure you do not let the beads dry out. 
c.     Resuspend beads in 500ml of low salt buffer. 
d.     Repeat steps a-c for a total of 3x low salt buffer washes, 3x high salt buffer washes, 2x LiCl buffer washes and 2x 1xTE washes.
SECTION: First elution, buffer exchange and secondary antibody incubation – Day 2
25. Elute washed complexes in 100 µL elution buffer containing fresh protease inhibitor cocktail for 30 min at 37 °C on a thermomixer. 30 min 

 

SECTION: First elution, buffer exchange and secondary antibody incubation – Day 2
26. During elution step prepare 3x Amicon Ultra buffer exchange columns by adding 500 µL Milli-Q H20 to columns and spinning at 14000 x g for 30 min at 4 °C. 
30 min 

SECTION: First elution, buffer exchange and secondary antibody incubation – Day 2
27. After chromatin elution, place samples on magnetic rack and wait for sample to turn clear. Move supernatant containing chromatin to a new low-bind tube.
SECTION: First elution, buffer exchange and secondary antibody incubation – Day 2
28. Take 10% volume (10 µL) from each chromatin IP as an in-line single ChIP control into a new low-bind tube, label and store at 4 °C until day 3.

SECTION: First elution, buffer exchange and secondary antibody incubation – Day 2
29. Bring each remaining chromatin sample up to 300 µL with ChIP buffer containing protease inhibitor cocktail.

SECTION: First elution, buffer exchange and secondary antibody incubation – Day 2
30. Decant the H20 from the prepared Amicon Ultra filters and add each chromatin sample to a separate filter.
SECTION: First elution, buffer exchange and secondary antibody incubation – Day 2
31. Spin at 14000 x g for 30 min at 4 °C.
30 min 

SECTION: First elution, buffer exchange and secondary antibody incubation – Day 2
32. Add 500 µL ChIP buffer containing protease inhibitor cocktail and spin at 14000g for 30 minutes at 4 °C.
30 min 

SECTION: First elution, buffer exchange and secondary antibody incubation – Day 2
33. Repeat steps 32 and 32.1 for a total of 2 washes.
SECTION: First elution, buffer exchange and secondary antibody incubation – Day 2
34. Recover as much chromatin sample as possible from within the Amicon Ultra filter device. (This is usually around 50 µL).

SECTION: First elution, buffer exchange and secondary antibody incubation – Day 2
35. Bring volume up to 500 µL with ChIP buffer containing protease inhibitors.

SECTION: First elution, buffer exchange and secondary antibody incubation – Day 2
36. Take antibody bound beads for the second incubation from 4 °C and place on magnetic rack.

SECTION: First elution, buffer exchange and secondary antibody incubation – Day 2
37. Wait for the solution to turn clear and remove the supernatant.
SECTION: First elution, buffer exchange and secondary antibody incubation – Day 2
38. Add appropriate chromatin samples to appropriate antibodies:

Add the IgG chromatin sample to the IgG-bead complexes.
Add the H3K4me3 chromatin to the H3K27me3-bead complexes.
Add the H3K27me3 chromatin to the H3K4me3-bead complexes.
SECTION: First elution, buffer exchange and secondary antibody incubation – Day 2
39. Incubate overnight at 4 °C with rotation.

SECTION: First elution, buffer exchange and secondary antibody incubation – Day 2
31.1. Carefully decant flowthrough and discard.
SECTION: First elution, buffer exchange and secondary antibody incubation – Day 2
32.1. Carefully decant flowthrough and discard.
SECTION: Second elution, de-crosslinking and DNA purification – Day 3
40. Wash chromatin-antibody-bead complexes a total of 9 times using the following steps keeping the samples cool by either placing magnet on ice or working in a cold room: 

a.     Place tubes on magnets rack and wait for solution to turn clear. 
b.     Carefully remove supernatant while on magnet making sure you do not disturb the beads. Make sure you do not let the beads dry out. 
c.     Resuspend beads in500 µLof low salt buffer. 
d.     Repeat steps a-c for a total of 3x low salt buffer washes, 3x high salt buffer washes, 2x LiCl buffer washes and 2x 1xTE washes.
SECTION: Second elution, de-crosslinking and DNA purification – Day 3
41. Elute complexes and reverse crosslinks in 100 µL elution buffer for a minimum of 2.5 hours up to overnight at 65 °C on a thermomixer. 150 min 

 


SECTION: Second elution, de-crosslinking and DNA purification – Day 3
42. Collect the 5% input sample (10 µL) and three in-line total control samples (10 µL each) from 4 °C storage. 

SECTION: Second elution, de-crosslinking and DNA purification – Day 3
43. Add 90 µL elution buffer to bring the final volume of each control to 100 µL.

SECTION: Second elution, de-crosslinking and DNA purification – Day 3
44. Place at 65 °C for 2.5 hours on a thermoshaker alongside the reChIP samples to de-crosslink. 150 min 

SECTION: Second elution, de-crosslinking and DNA purification – Day 3
45. After de-crosslinking, place all tubes on the magnetic rack. 
You should have a total of 7 tubes corresponding to input, 3x in-line total controls and 3x reChIPs. 

SECTION: Second elution, de-crosslinking and DNA purification – Day 3
47. Add 2 µL RNaseA (NEB) to each tube and incubate at 37 °Cfor 30 min. 30 min 

SECTION: Second elution, de-crosslinking and DNA purification – Day 3
48. Add 2 µL Proteinase K (NEB) to each sample and incubate at 37 °C for 1 hour. 60 min 

SECTION: Second elution, de-crosslinking and DNA purification – Day 3
49. Purify DNA using Ampure beads. 

a.     Bring Ampure beads to room temperate prior to use. 
b.     Add beads to sample in a 1:1.8 ratio (e.g. 180 µL beads to 100 µL sample) and pipette up and down to mix. 
c.     Incubate at room temperate for 5 minutes. 5 min 
d.     Place tubes on magnetic rack for 5 mins and remove supernatant. 5 min 
e.     While tubes are on the rack wash the beads with 400 µLof freshly prepared 80 % (v/v) ethanol in molecular grade water, remove ethanol leaving only bead-bound DNA. 
f.      Repeat step e for a total of 2 washes. 
g.     While the tubes are on the magnet, allow beads to air dry for up to 5 minutes. Note – it is important not to over dry here. Proceed to DNA elution before the beads start to crack. 
h.     To elute DNA, remove tubes from the magnetic rack and resuspend beads in Xml (see step 50 below) of 10 millimolar (mM) Tris-HCL pH8.0. 
i.      Incubate at room temperature for 5 minutes. 5 min 
j.      Place tubes back on the magnetic rack for 5 minutes and transfer DNA solution to new tube. 5 min 

SECTION: Second elution, de-crosslinking and DNA purification – Day 3
50. For downstream qPCR analysis elute in 60-80 µL 10 millimolar (mM) Tris-HCL pH8.0 and use 1 µL per qPCR reaction.

SECTION: Second elution, de-crosslinking and DNA purification – Day 3
51. For downstream NGS elute in 20 µL 10 millimolar (mM) Tris-HCL pH8.0. 
a.     Use 1 µL of eluate to determine the DNA concentration using a Qubit fluorometer. 
b.     Use 2 µLfor qPCR analyses of positive and negative control regions (dilute 4x to give final volume  of 8 µL and use 1 µL per reaction). 
c.     Use remaining eluate (17 µL) to prepare libraries using NEBNext® Ultra™ II DNA Library Prep Kit or similar following manufacturer’s instructions.

SECTION: Second elution, de-crosslinking and DNA purification – Day 3
46. When the solution turns clear move supernatant to new low-bind tubes.