Feb 22, 2024

Public workspaceCHIP-reCHIP Protocol for Mapping Bivalent Chromatin

DOI
dx.doi.org/10.17504/protocols.io.8epv5x815g1b/v1
  • Janith A Seneviratne1,2,
  • William W H Ho3,
  • Eleanor Glancy1,2,
  • Melanie A Eckersley-Maslin1,2,4
  • 1Peter MacCallum Cancer Centre, Melbourne, Victoria, 3000, Australia;
  • 2Sir Peter MacCallum Department of Oncology, The University of Melbourne, Victoria, 3010, Australia;
  • 3Peter MacCallum Cancer Centre, Melbourne, Victoria, 3000, Australia.;
  • 4Department of Anatomy and Physiology, The University of Melbourne, Victoria, 3010, Australia.
  • Melanie A Eckersley-Maslin: melanie.eckersley-maslin@petermac.org;
eckmaslab
Open access
DOI: dx.doi.org/10.17504/protocols.io.8epv5x815g1b/v1
External link: http://10.1186/s13072-024-00527-9
Protocol Citation: Janith A Seneviratne, William W H Ho, Eleanor Glancy, Melanie A Eckersley-Maslin 2024. CHIP-reCHIP Protocol for Mapping Bivalent Chromatin. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5x815g1b/v1
Manuscript citation:
Seneviratne JA, Ho WWH, Glancy E, Eckersley-Maslin MA. A low-input high resolution sequential chromatin immunoprecipitation method captures genome-wide dynamics of bivalent chromatin. Epigenetics Chromatin. 2024 Feb 10;17(1):3. doi: 10.1186/s13072-024-00527-9. PMID: 38336688; PMCID: PMC10858499.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working. Some steps (noted) may require optimisation for different cell lines.
Created: February 01, 2024
Last Modified: February 22, 2024
Protocol Integer ID: 94529
Keywords: Bivalency, Bivalent chromatin, ChIP-reChIP, Chromatin immunoprecipitation, Embryonic stem cells, Epigenetics, H3K27me3, H3K4me3, Plasticity, Sequential ChIP
Funders Acknowledgement:
Snow Medical
Grant ID: SF2020-52
Abstract
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).
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
Reagent or ResourceSourceCatalogue No.
16% Formaldehyde (w/v), Methanol freeLife Technologies28908
GlycineSigma G8898
Sodium deoxycholateSigma30970
Lithium Chloride solution 8MSigmaL7026
MNaseNEBM0247S
RNaseANEBT3018-2
ProteinaseKNEBP8107S
Protein A DynaBeadsThermo Fisher10002D
Protein LoBind 1.5ml tubesEppendorf0030108442
Magnetic RackInvitrogen12321D
Protease Inhibitor cocktailRoche05892791001
Amicon UltraMilliporeMPUFC5003BK
NEBNext Ultra II DNA Library Prep Kit NEBE7645L

Protocol materials
ReagentNEBNext Ultra II DNA Library Prep Kit for Illumina - 96 rxnsNew England BiolabsCatalog #E7645L
Step 1
ReagentLithium Chloride solution 8MMerck MilliporeSigma (Sigma-Aldrich)Catalog #L7026
Step 1
ReagentProteinaseKNew England BiolabsCatalog #P8107S
Step 1
ReagentSodium deoxycholateMerck MilliporeSigma (Sigma-Aldrich)Catalog #30970
Step 1
ReagentMNaseNew England BiolabsCatalog #M0247S
Step 1
ReagentProtease Inhibitor cocktailRocheCatalog #05892791001
Step 1
Reagent16% Formaldehyde (w/v), Methanol freeLife TechnologiesCatalog #28908
Step 1
ReagentProtein A DynaBeadsThermo Fisher ScientificCatalog #10002D
Step 1
ReagentGlycineMerck MilliporeSigma (Sigma-Aldrich)Catalog #G8898
Step 1
ReagentRNAse ANew England BiolabsCatalog #T3018-2
Step 1
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
ReagentFinal ConcentrationAmount in 100ml
1M Tris pH7.410mM1ml
Sorbitol1M18.217g
1M NaCl50mM5ml
1M MgCl25mM0.5ml
1M CaCl21mM0.1ml
IGEPAL0.075%75ml
ddH2O Up to 100 ml

CHROMATIN PRECIPITATION BUFFER
ReagentFinal ConcentrationAmount in 500ml
1M Tris pH7.420mM10ml
0.5M EDTA2mM2ml
1M NaCl150mM75ml
Triton X1000.1%0.5ml
ddH2O Up to 500ml
LOW SALT WASH BUFFER
ReagentFinal ConcentrationAmount in 500ml
1M Tris pH820mM10ml
0.5M EDTA2mM2ml
5M NaCl150mM15ml
Triton X1001%5ml
10% SDS0.1%5ml
ddH2O Up to 500ml

HIGH SALT WASH BUFFER
ReagentFinal ConcentrationAmount in 500ml
1M Tris pH820mM10ml
0.5M EDTA2mM2ml
5M NaCl500mM50ml
Triton X1001%5ml
10% SDS0.1%5ml
ddH2O Up to 500ml

LiCl WASH BUFFER
ReagentFinal ConcentrationAmount in 500ml
8M LiCl250mM15.625ml
IGEPAL1%5ml
10% deoxycholate1%50ml
0.5M EDTA1mM1ml
1M Tris pH7.410mM5ml
ddH2O Up to 500ml

ELUTION BUFFER
ReagentFinal ConcentrationAmount in 10ml
1M Tris pH7.410mM0.1ml
0.5M EDTA1mM0.02ml
10% SDS1%1ml
ddH2O Up to 10ml
Key Resources & reagents
Key Resources & reagents
Reagent16% Formaldehyde (w/v), Methanol freeLife TechnologiesCatalog #28908
ReagentGlycineMerck MilliporeSigma (Sigma-Aldrich)Catalog #G8898
ReagentSodium deoxycholateMerck MilliporeSigma (Sigma-Aldrich)Catalog #30970
ReagentLithium Chloride solution 8MMerck MilliporeSigma (Sigma-Aldrich)Catalog #L7026
ReagentMNaseNew England BiolabsCatalog #M0247S
ReagentRNAse ANew England BiolabsCatalog #T3018-2
ReagentProteinaseKNew England BiolabsCatalog #P8107S
ReagentProtein A DynaBeadsThermo Fisher ScientificCatalog #10002D
ReagentProtease Inhibitor cocktailRocheCatalog #05892791001
ReagentNEBNext Ultra II DNA Library Prep Kit for Illumina - 96 rxnsNew England BiolabsCatalog #E7645L

Equipment
Amicon Ultra-0.5 Centrifugal Filter Unit
NAME
Centrifugal Filter Unit
TYPE
Millipore
BRAND
UFC5003BK
SKU
https://www.merckmillipore.com/GB/en/product/Amicon-Ultra-0.5-Centrifugal-Filter-Unit,MM_NF-UFC5003BK
LINK

Equipment
Protein Lobind 1.5mL tubes
NAME
Tubes
TYPE
Eppendorf
BRAND
0030108442
SKU
https://www.eppendorf.com/au-en/eShop-Products/Laboratory-Consumables/Tubes/Protein-LoBind-Tubes-p-0030108116
LINK
1.5mL, snap-cap, Protein LoBind, PCR clean, colourless
SPECIFICATIONS

Equipment
DynaMag-2
NAME
Magnet
TYPE
Invitrogen
BRAND
12321D
SKU
https://www.thermofisher.com/order/catalog/product/12321D#/12321D
LINK

Chromatin Fixation
Chromatin Fixation
2h

Note
Before you start: pre-warm DMEM; pre-cool centrifuge to 4 degrees; add Roche cOmplete EDTA-free protease inhibitor tablets to PBS/EDTA and keep on ice until required.

 

Note
Note: all steps must be performed on ice or at 4 degrees unless otherwise specified.



Note
Note: chromatin fixation can also be performed on cells in suspension by resuspended a pellet of a known number of washed live cells directly into the 1% formaldehyde solution and incubating for 8 minutes on a rocker, quenching with glycine as below and then mild centrifugation to get a fixed cell pellet( rather than scraping cells).

Grow cells until they are 70-80% confluent across several plates. One will be used for counting and the remaining for fixation (collection plates).
Collect and count cells on the counting plate: Remove media from the counting plate and wash once with DPBS.
Add Amount1 mL of Trypsin or appropriate dissociation reagent per 10cm plate and incubate until cells lift off plate as single cells.

Quench trypsin with Amount5 mL of media.

Take aliquot and count cells to determine total cell number per plate and therefore the total number of cells overall.
Prepare Concentration1 %(w/v) formaldehyde solution by adding Amount0.625 mL of Concentration16 %(w/v) formaldehyde solution to Amount9.375 mL of prewarmed DMEM per 10cm collection plate.

Note
Scale up volumes if you have more than one 10cm collection plate.


Remove media and wash cells on collection plates with DPBS.
Cross link cells by adding Amount10 mL of Concentration1 %(w/v) formaldehyde solution per plate of adherent cells for 8 min at room temperature.

Quench with 1ml Concentration1 Molarity (M) Glycine per plate of adherent cells to a final concentration Concentration125 millimolar (mM) , for 5 min at room temperature.

Pour off medium and wash cells with Amount10 mL of ice cold DPBS.

Pour off DPBS, scrape cells in residual PBS and transfer to low-bind 1.5ml tube. Pool cells from all plates into one tube.
Centrifuge for at 500xg at 4 degrees for 10 minutes Centrifigation500 x g, 4°C, 00:10:00 .

10m
Remove supernatant and resuspend cells to 2x10^7 cells per 1ml in PBS/5mM EDTA containing protease inhibitors. Aliquot by adding Amount0.1 mL cell slurry (corresponding to 2x10^6 cells) per low-bind tube.

Pellet aliquots by centrifugation at 500xg at 4 degrees for 5 minutes Centrifigation500 x g, 4°C, 00:05:00 .

5m
Remove supernatant and snap freeze on dry ice or in liquid nitrogen. Store pellets at Temperature-80 °C for up to 6 months .

Chromatin fragmentation and primary antibody incubation - Day 1
Chromatin fragmentation and primary antibody incubation - Day 1
1d 2h

Note
Before you start: pre-cool centrifuge; add Roche cOmplete EDTA-free protease inhibitor tablets to ChIP buffer and NP buffer and keep on ice until needed.

Note
Note: Unless otherwise specified all procedures should be carried out between 2-8 degrees 


Note
Note: NP buffer composition and cell lysis conditions may need to be optimised depending on your cell type.

Note
Note: volumes below are to process one sample corresponding to one vial of 2x10^7 cells and will result in input, in-line H3K4me3, in-line H3K27me3, IgG-IgG reChIP and bivalent K4-K27 and K27-K4 reChIP samples that can be further processed by qPCR and/or library preparation for sequencing. If you have more than one sample (e.g. biological replicate or other condition), scale volumes accordingly. We typically do not process more than 4 samples at any time.
Note: sonication can be used instead of MNase digestion. We routinely use both fragmentation methods in performing reChIP experiments with similar results.

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 Amount150 µL . This corresponds to:

  • Amount20 µL of beads to preclear 2x10^6 cells.
  • Amount120 µL of beads for antibody-complex formation (Amount20 µL per antibody-complex of which there are 6 reactions in total: 2xIgG, 2x H3K27me3, 2xH3K4me3)
  • Amount10 µL for pipetting errors

Take Amount150 mL Protein A dynabeads, place on magnetic rack and remove supernatant. 

Remove tube from rack and resuspend in Amount500 µL of 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 Amount150 µL of cold ChIP buffer.
     Keep chilled on ice until needed later.

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)
Add Amount500 µL of cold ChIP buffer to each tube.

Add Amount20 µL of pre-washed Protein A dynabeads to each tube.

Add appropriate antibody to each tube - Amount1 µg IgG (Invitrogen), Amount10 µL H3K27me3 (CST 9733), Amount2 µL H3K4me3 (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.

Incubate bead/antibody mix at Temperature4 °C on a rotator for at least 3 hours Duration03:00:00 .

3h
Chromatin preparation – 1 hour preparation time plus at least 3 hours incubation
4h
Thaw 1 vial of 2x10^6 crosslinked cells on ice
Resuspend cell pellet (2x10^6 cells) in Amount97.68 µL NP buffer supplemented with Amount0.7 µL of Concentration55 millimolar (mM) beta-mercaptoethanol, Amount1.82 µL of Concentration0.1 Molarity (M) spermidine and freshly added protease inhibitor cocktail.

Fragment chromatin with MNase: prepare MNase master mix (Amount20 µL per sample)
  • Amount12 µL 10X MNase buffer
  • Amount1.76 µL 100mM DTT 
  • Amount3.84 µL dH20
  • Amount2.4 µL MNase




Add Amount20 µL  of MNase master mix to each tube of 2x10^6 cells in NP buffer.

Incubate at Temperature37 °C with shaking at Shaker600 rpm for 7.5 - 15 min.

Note
Note: This amount of MNase and digestion time will need to be titrated for each cell line. These conditions have been optimised to yield predominantly mono nucleosomal DNA for 2x10^6 mouse embryonic stem cells. If preferred sonication can be used to fragment chromatin instead. 

15m
During digestion prepare STOP buffer: 
  • Amount15 µL Concentration100 millimolar (mM) EDTA
  • Amount15 µL Concentration1 % (v/v) Triton / Concentration1 % (v/v) deoxycholate solution
per sample

Add Amount26.4 µL of STOP buffer to each sample to stop the MNase digestion.
Incubate on ice for 5 minutes. Duration00:05:00

5m
Vortex each tube for 30 seconds each.
Bring volume up to Amount600 µL by adding Amount473.6 µL cold ChIP buffer containing protease inhibitor cocktail.

Add Amount20 µL of pre-washed protein A Dynabeads from step 19 to the chromatin and incubate for at least 3 hours at Temperature4 °C on rotator to pre-clear the chromatin. This is critical to reduce non-specific binding and decrease background signal. Duration03:00:00

3h
Overnight incubation with primary antibody – 30 min and overnight
1d 0h 30m
Take all bead-antibody tubes from the 4-degree rotator.
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.

Note
We recommend sitting the magnetic rack on ice to keep cool during these steps. Alternatively, these steps can be performed in a cold room.

The remaining bead-antibody mixes can be stored at 4 degrees until needed on day 2.
Take Amount10 µL of pre-cleared chromatin supernatant to a separate tube and label as 5% input control. Keep at Temperature4 °C until day 3.

Remove the supernatant from the antibody-bead complexes on the magnetic rack and discard.
Add Amount200 µL of the pre-cleared chromatin supernatant to each antibody/bead mixture.

Top up each chromatin/antibody/bead mixture with Amount300 µL of ChIP buffer to final volume of Amount500 µL . 

Incubate DurationOvernight at Temperature4 °C on a rotator.

15m
First elution, buffer exchange and secondary antibody incubation – Day 2
First elution, buffer exchange and secondary antibody incubation – Day 2
1d 2h

Note
Before you start: Prepare 30ml of ChIP buffer containing protease inhibitor cocktail. Prepare 5ml of elution buffer containing protease inhibitor cocktail; pre-cool centrifuge to 4 degrees.


Collect chromatin-antibody-bead samples from overnight Temperature4 °C rotation.

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.
Elute washed complexes in Amount100 µL elution buffer containing fresh protease inhibitor cocktail for 30 min at Temperature37 °C on a thermomixer. Duration00:30:00

Note
If you do not have access to a shaking heat block, gently flick the tubes periodically during the incubation to ensure beads remain suspended in solution.

30m
During elution step prepare 3x Amicon Ultra buffer exchange columns by adding Amount500 µL Milli-Q H20 to columns and spinning at Centrifigation14000 x g for 30 min at Temperature4 °C .
Duration00:30:00

30m
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.
Take 10% volume (Amount10 µL ) from each chromatin IP as an in-line single ChIP control into a new low-bind tube, label and store at Temperature4 °C until day 3.

Bring each remaining chromatin sample up to Amount300 µL with ChIP buffer containing protease inhibitor cocktail.

Decant the H20 from the prepared Amicon Ultra filters and add each chromatin sample to a separate filter.
Spin at Centrifigation14000 x g for 30 min at Temperature4 °C .
Duration00:30:00

30m
Carefully decant flowthrough and discard.
Add Amount500 µL ChIP buffer containing protease inhibitor cocktail and spin at 14000g for 30 minutes at Temperature4 °C .
Duration00:30:00

30m
Carefully decant flowthrough and discard.
Repeat steps 32 and 32.1 for a total of 2 washes.
Recover as much chromatin sample as possible from within the Amicon Ultra filter device. (This is usually around Amount50 µL ).

Bring volume up to Amount500 µL with ChIP buffer containing protease inhibitors.

Take antibody bound beads for the second incubation from Temperature4 °C and place on magnetic rack.

Wait for the solution to turn clear and remove the supernatant.
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.
Incubate overnight at Temperature4 °C with rotation.

Second elution, de-crosslinking and DNA purification – Day 3
Second elution, de-crosslinking and DNA purification – Day 3
4h
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 inAmount500 µL 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.
Elute complexes and reverse crosslinks in Amount100 µL elution buffer for a minimum of 2.5 hours up to overnight at Temperature65 °C on a thermomixer. Duration02:30:00

Note
Note: no protease inhibitors are required for this elution step.


2h 30m
Collect the 5% input sample (Amount10 µL ) and three in-line total control samples (Amount10 µL each) from Temperature4 °C storage.

Add Amount90 µL  elution buffer to bring the final volume of each control to Amount100 µL .

Place at Temperature65 °C for 2.5 hours on a thermoshaker alongside the reChIP samples to de-crosslink. Duration02:30:00

2h 30m
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. 

When the solution turns clear move supernatant to new low-bind tubes.
Add Amount2 µL RNaseA (NEB) to each tube and incubate at Temperature37 °C for 30 min. Duration00:30:00

30m
Add Amount2 µL Proteinase K (NEB) to each sample and incubate at Temperature37 °C for 1 hour. Duration01:00:00

1h
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. Amount180 µL beads to Amount100 µL sample) and pipette up and down to mix.
c.     Incubate at room temperate for 5 minutes. Duration00:05:00
d.     Place tubes on magnetic rack for 5 mins and remove supernatant. Duration00:05:00
e.     While tubes are on the rack wash the beads with Amount400 µL of freshly prepared Concentration80 % (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 Concentration10 millimolar (mM) Tris-HCL pH8.0.
i.      Incubate at room temperature for 5 minutes. Duration00:05:00
j.      Place tubes back on the magnetic rack for 5 minutes and transfer DNA solution to new tube. Duration00:05:00

20m
For downstream qPCR analysis elute in Amount60-80 µL Concentration10 millimolar (mM) Tris-HCL pH8.0 and use Amount1 µL per qPCR reaction.

For downstream NGS elute in Amount20 µL Concentration10 millimolar (mM) Tris-HCL pH8.0.
a.     Use Amount1 µL of eluate to determine the DNA concentration using a Qubit fluorometer.
b.     Use Amount2 µL for qPCR analyses of positive and negative control regions (dilute 4x to give final volume  of Amount8 µL and use Amount1 µL per reaction).
c.     Use remaining eluate (Amount17 µL ) to prepare libraries using NEBNext® Ultra™ II DNA Library Prep Kit or similar following manufacturer’s instructions.

Protocol references
Seneviratne JA, Ho WWH, Glancy E, Eckersley-Maslin MA. A low-input high resolution sequential chromatin immunoprecipitation method captures genome-wide dynamics of bivalent chromatin. Epigenetics Chromatin. 2024 Feb 10;17(1):3. doi: 10.1186/s13072-024-00527-9. PMID: 38336688; PMCID: PMC10858499.