Wagner lab simplified chromatin immunoprecipitation (ChIP)

Introduction: 

This simplified ChIP protocal can be used for transcription factors or chromatin regulators.

Procedure : 

I. Harvest plant tissue for crosslinking: 

  • Harvest 300mg seedlings into a 25ml glass Scintillation vial containing 10 ml of 1X PBS solution (no need to sit).
  • Harvest 300mg young inflorescences into a 25ml Scintillation vial containing 10 ml of 1X PBS solution and let the inflorescences sit in the PBS solution for 2-3hours.
  •  Remove 1X PBS with 10ml pipette and replace with 10ml room temperature 1% formaldehyde solution in 1X PBS (270ul 37% formaldehyde per 10ml 1X PBS) (to cross-link tissue).
  • Vacuum infiltrate tissue until it sinks (2 vacuums for seedlings, usually 3 to 4 for inflorescences), not exceeding 15min total exposure time to formaldehyde.  Typical seedling vacuum:
    • Preincubate in hood: 4 min
    • First vacuum: 2-3 min, or stop when bubbles start to boil over
    • Vacuum release
    • Second vacuum: 2-3 min – bubbles should slow down by the end of vacuum time
    • Vacuum release Seedlings should sink to the bottom after vacuum release.  If still

floating, vacuum again.

  •  
    • Post-incubate in hood: ~ 3 min
  • Remove formaldehyde in hood with 10ml pipette and add 10ml 0.125 M room temperature glycine solution (to quench cross-linking reactions).  Vacuum for once for ~1.5 min. The total glycine incubation time is 5 min.
  • Remove glycine in hood and rinse 2 times with 10 ml cold 1x PBS solution on bench (PBS should be cold at this point to preserve cross-links).
  • Dry plant seedlings briefly (blot on paper towel), weigh (should be 200-300mg), and insert tissues into 2ml tubes
  • Freeze in liquid nitrogen and store at –80˚C

 

II. Preparation of magnetic beads

Note:  For all steps involving magnetic beads, spin first briefly to collect the beads, let tubes sit on magnetic stand for 1 min to allow beads to migrate, then remove supernatant.

  • Make fresh PBS/BSA Solution (5.0 ml per sample).
  • For two samples (e.g. dexamethasone and mock-treated), transfer 100 µl of Dynal Protein A magnetic beads into two 2 ml tubes (100 µl/ 2ml tube).  Note:  Be sure to vortex the Protein A beads well (1-2min) before aliquotting.
  • Remove the solution using a magnetic stand.
  • Resuspend in 1 ml of PBS with 5mg/ml BSA and rotate in cold room at 16 rpm for 10 min.
  • Repeat this washing one more time.
  • Resuspend beads in 1 ml PBS/BSA and add 80ul LFY antibody (3M7 or 3M9), 5 ul GFP antibody or 10 ul HA antibody to one of the two tubes.  (The second tube will be used for preclearing of the two samples.)
  • Incubate with rotation at 4°C O/N.
  • Thaw Nuclei Extraction Buffer (3.0ml per sample) and store remaining PBS/BSA at 4C O/N.

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Next day:

  • Briefly centrifuge and remove the PBS/BSA using a magnetic stand.
  • Resuspend beads in 1ml PBS/BSA and incubate on rotator at 4°C for 10 min.
  • Repeat washing with 1ml of PBS/BSA.

·       Carefully remove all liquid, and resuspend in 60 µl of PBS/BSA per tube.   III. Protein-DNA extraction

  • Add Protease Inhibitors (from 100X stock) and β-Me (3.12ul/ml) to Nuclei Extraction Buffer.  Make 3.0 ml per sample.
  • Grind samples to a fine powder using mortar and pestle, keeping the tissue frozen throughout.
  • Add 2.5ml nuclei extraction buffer with protease inhibitors and ß-ME and keep grinding until thawed.
  • Set up small funnel over 50ml Falcon tube with two layers of Miracloth in funnel. Pass filtrate through the Miracloth 2 times and transfer filtrate to a 2ml tube.  Be sure to squeeze the Miracloth after second pass-thru to extract all of the liquid. (Change gloves after each sample.) Keep samples on ice until all are ground.
  • Spin for 5min at 10,000g (rcf scale) at 4°C
  • Remove supernatant and resuspend pellet in 75µl nuclei lysis buffer – this will require stirring with pipette tip. Note: Try to avoid foaming.
  • Leave sitting on ice for 30 min with occasional stirring
  • Bring final volume up to 700µl with ChIP dilution buffer without Triton (add 625ul), and add 200µl glass beads.  Note:  You can measure the glass beads by filling an upside-down 200ul pipet tip.

 

IV. IP

Sonication

  • Sonicate seven times for 10s each (~ P3.2) with Fisher Dismembrator (the size range of sheared DNA should be around 200-700bp).  Let sample sit on ice for 50 sec between sonications to allow it to cool.  Note:  If samples get too hot, the proteins will be denatured.
  • Transfer the sonicated solution to a new 2ml tube using a very small pipet tip.
  • Typically 665ul solution after sonication. Add 200ul ChIP dilution buffer w/Triton and 35ul of 22% Triton X-100 to attain 900µl of sample with 1.1% triton X-100 concentration (to limit protease activity).     
  • Spin full 13,200 rpm for 5min at 4°C.
  • Remove from centrifuge immediately, and transfer clean supernatant to a new 2ml tube.  

Preclearing

  • Add 30 µl of BSA-coated beads from III per tube.
  • Incubate for 1 hr at 4 °C with rotation.
  • Transfer cleared solution to a new 2ml tube using magnet.
  • Remove 2/100th (18µl) as 2% INPUT sample.  Note:  Remove 2%, not 1%, so that concentration can be measured later.

IP

  • Add 2 µg of sheared salmon sperm DNA and 30 µl of antibody/BSA-coated beads per tube.
  • Leave rotating overnight at 4°C

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Next day

 

V. Washes

  • Remove supernatant from beads and reserve for later verification of sonication. 

Note: Rotate all washes for 25 min at RT except for TE (10 min)

  • Wash 2X with cold 1ml Low Salt Wash buffer
  • Wash 2X with cold 1ml High Salt Wash buffer
  • Wash 2X with cold 1ml 250mM LiCl buffer
  • Wash 2X with cold 1ml .5X TE
  • Pipette off all liquid and add 50µl elution buffer (Nuclei Lysis Buffer).
  • Incubate 25min at 65°C in large incubator to separate sample from beads
  • Remove and store elution buffer, add another 50µl elution buffer to beads and repeat heating and suspension.
  • Combine both sets of elution buffer

·       Discard beads

 

VI. X-link reversal

  • Add 82ul nuclei lysis buffer to reserved Input samples.
  • Add 6 µl of 5M NaCl to Input and ChIP samples.
  • Reverse X-link overnight at 65°C

 

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Next day:   VII. DNA cleanup procedure

Use Qiagen PCR purification Kit to purify samples.

  • Add 5 volumes (550ul) Buffer PB to each tube.   

Note:  Do not use Buffer PBI if using the ChIP sample for microarray hybridization.

  • Shake samples for 30 min at room temperature. 
  • Load QIAquick spin columns with samples and centrifuge for 1 min at 13.2k rpm. 
  • Reload flow-thru and spin again. 
  • Discard flow-thru. 
  • Add 750ul Buffer PE to column and centrifuge 1 min at 13.2k rpm.   
  • Discard flow-thru and spin again 1 min at 13.2 rpm. 
  • Place column in a clean 1.5ml tube and add 100ul .5X EB Buffer to center of column. Let sit 5 min.  
  • Centrifuge at 13.2K rpm 1 min. 
  • Reload flow-thru, let sit 5 min, and spin again for 1 min. 

 

Reagent

Vendor

Catalog #

BSA (Initial fractionation by heat shock, Fraction V)

Sigma

A-7906

Glass beads, 1.0mm

BioSpec Products, Inc.

11079110

Dynal Protein A beads

Invitrogen

100-02D

Miracloth

Calbiochem

475855

β-Me

Sigma

M-3148

Sheared Salmon Sperm DNA

 

 

Protease Inhibitors, aliquotted and stored at -20C

Sigma

P9599-5ml

Magnetic Stand

Promega

 

QIAQUICK PCR Purification Kit

QIAGEN

28104

Gel Loading Tips, 1-200ul

USA Scientific

1022-0600

 

References

Kwon, C.S., Chen, C., and Wagner, D. (2005). WUSCHEL is a primary target for transcriptional regulation by SPLAYED in dynamic control of stem cell fate in Arabidopsis. Genes Dev 19, 992-1003.

Yamaguchi, A., Wu, M.F., Yang, L., Wu, G., Poethig, R.S., and Wagner, D. (2009). The  microRNA-regulated SBP-Box transcription factor SPL3 is a direct upstream activator of LEAFY, FRUITFULL, and APETALA1. Developmental Cell 17, 268-278.

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