Chewbby

Sonicated Chromatin Immunoprecipitation from Yeast

 

(Adapted from Liu et al. PLoS Biol 2005 3(10):1753-1769)

(Adapted from single-nucleosome resolution ChIP from yeast V3.0 adapted for magnetic beads by Filleton F.)

(Adapted from Pascal Bernard team)

1 unit of DO600 is around 1.107 cells per mL. For ChIP, it is better to collect cells in log phase (between 0.8 to 1.4) but if blue light illumination is necessary before ChIP collect cells in stationary phase just after illumination.

Step 1 : Culture and collection of the yeast cells

Equilibrate protA-sepharose beads if necessary

Inoculate a culture of 120 mL SD-MET-TRP medium with a single colony (in a 1L flask). Grow overnight at 30°C.

Prepare glycine 2,5M (9,37g in 50ml distilled water), cold TBS 1x and cool the centrifuge at 4°C

[Optional : Blue light illumination : Illuminate 55 to 60 mL of overnight culture into a 6 well plate with SPOT (intensity 255, 450nm, pulse 1secON/9sOFF during 1 h, at 30°C). Keep the other 50 mL half of culture in the dark.]

Take OD600 of the samples. Transfer 50 mL of the culture to a Flacon 50 mL and add 1.4 mL of formaldehyde 37% (Sigma F8775) ## FUME HOOD ## (final concentration 1%)

Incubate 15 min at room temperature on rotating rods.

Add, under fume hood, 2.5 mL of glycine 2,5M (fresh prepared, role : quinch the cross linker).

Invert gently and incubate 5 min at room temperature on rotating rods

Centrifuge for 5 min at 4000 rpm (~3000g) at 4°C on Falcon 50 mL tubes on Allegra.

Pour the supernatant in hazardous waste ## FUME HOOD ## (containing formaldehyde).

Wash twice with 30 ml of cold TBS 1x, mix by inverting and centrifuge for 5 min and 3000g at 4°C (remove cross-linker) (trash supernatant in the sink).

Make aliquot of 1.108 cells/tube and pellet the cells (Sarstedt ref 72.693.005).

Proceed directly to step 2 or snapFreeze in liquid N2 and store at -80°C.

Step 2 : Lysis of the yeast cells (Precellys)

Prepare Lysis buffer w/o Protease Inhibitor Cocktail

Thaw aliquot of 1.108 cells/tube on ice. Resuspend with 300 µL of Lysis Buffer + Protease Inhibitor Cocktail. Mix well by pipetting.

Add ~ 500 µL cold “acid-washed” beads (Sigma G8772) until the meniscus.

On PreCellys machine : disrupt cells during 2 cycles of 2*10 secondes (with 10 secondes pause) at 6800 rpm, put tubes on ice during 1 min between the 2 cycles.

Recovery of the lysate : Put the tube upside down, lysate and beads will go down in the cap. Pierce the bottom of the tube with a blue needle (23G) heated to white. Return the pierced tube into a hemolysis tube. Spin down 1 min, 2500 rpm at 4°C. Resuspend lysate. Keep tubes on ice. Verify lysis on microscope.

Step 3 : Sonication of the chromatine (Covaris)

Transfer the lysate into Covaris tube (qsp 1 mL Lysis Buffer).

Prepare the Covaris machine (instructions next to it). Fill the tank until “Fill 10”. Water level must be 1 mm under the cap of the tube.

Covaris program : P 140 W, duty factor 5%, 200 burst per cycle, 15 min.

Spin down lysate 10 min, 14000 rpm, 4°C. Transfer in a new tube. Spin down 5 min, 14000 rpm, 4°C. Transfer supernatant in a new tube, complete with Lysis buffer to have a total volume of 1 mL. Vortex. (1 mL = 200 µL for “Quality Control”, 750 µL for “IP” and “50 µL for “INPUT”)

[If necessary : withdraw 200 µL of sample and SnapFreeze the rest in liquid N2 and store at -80°C. Check sonication on 2% agarose gel using the 200 µL you drew (see Sonication Quality Control).

Sonication Quality Control [by phenol/chloroform extraction] :

  1. Add 200 µL Elution Buffer Shearing, incubate 15 to 30 min at 65°C
  2. Add 400 µL Phenol/chloroforme/IAA, vortex ++++
  3. Spin down 10 min, 14000 rpm, at room temperature.
  4. Transfer 270 µL in a new tube, add 30 µL NaCl 2M and 4 µL Glycogen 20 mg/mL. Vortex
  5. Add 750 µL Ethanol 100 %, vortex. Keep 30 min at -20°C
  6. Spin down 30 min, 14000 rpm, at 4°C. Remove supernatant.
  7. Wash with 500 µL EtOH 70%, spin down 5 min, 14000 rpm, at 4°C. Remove supernatant.
  8. Dry 5 to 10 min at 37 °C.
  9. Resuspend with 15 µL TE8 + RNase A 1 mg/mL final.
  10. Incubate 30 min at 37°C (mix several time).
  11. Add 3 µL Loading dye 6X and run on a 2% long agarose gel with an appropriate DNA sizing ladder (1 h at 100 V or 2h at 50V)

Sonication Quality Control [with the Tape Station] : TO DEVELOP AND TEST

  1. Take 50 µL of “Quality Control”and add 1 µL RNaseA (10-15 min at 37°C)
  2. Add 2 µL NaCl 8M + 2 µL Protease K (30 min at 65°C)
  3. Purify with PCR Clean-up Columns (Macherey Nagel), elute with 40 µL.
  4. Use 1 or 2 µL to control on Tape Station (HS D1000 kit)

 

Step 4a: Dynabeads pA preparation (Life Technologies 10002D, stored at 4°C)

[For IP : Predict 35 µL of beads pA and X µL of antibody (depends on the antibody manual) and the same amount of beads without antibody (for Input fraction). Vortex manualy the beads stock solution before each pipeting.]

At room temperature with sterile gloves :

Transfer the required volume of beads in a 1.5 mL Low Binding Microcentrifuge Tube. Put the tube on the magnetic stand until the sample appears clear.

Gently remove and discard clear sample taking care not to disturb beads.

Remove the tube from magnetic stand and resuspend beads with 500 µL of Lysis Buffer (+Protease Inhibitor cocktail). Repeat the previous step 2 other times to wash the bead (total of 3 washings).

Resuspend 1 volume of the beads (35 µL) in 1 volume of Lysis Buffer (+Protease Inhibitor cocktail) (for several IPs add 1 volume of beads). Split beads in 2, one tube for IP, one for Input.

Add in the IP tube the corresponding quantity of antibody (for several IPs add 1 volume of antibody) (ex : Add 2 µL (4 ug) per IP of anti-V5-TAG antibody (BioRad MCA1360)). Incubate beads on rotating wheel at room temperature for 20 minutes to 2 hours.

Step 4b: ImmunoPrecipitation

In a new Low Binding Microcentrifuge Tube, transfer 750 µL of the remaining supernatant obtain by sonication (“IP” tube).

Add 35 µL of previously prepared Dynabeads pA preparation (Beads +antibody). [Vortex manually the beads before each pipeting.]

Incubate the final “IP” overnight on a rotating wheel at 4°C.

Incubate 50 µL of the “INPUT” fraction at 4 °C overnight on a rack.

At morning, add 100 µL of Lysis buffer to the 50 µL of the “INPUT” samples. Then add 35 µL of previously prepared Dynabeads pA WITHOUT antibody. Incubate 1 hour on rotating wheel at 4°C.

During this incubation : put “IP” tubes and proceed to the washing on the magnetic stand until the magnetic beads appears magnetized. Don't panic : during the ~ 3/4 first washing the solution is hardly clear !!! Gently remove and discard clear sample taking care not to disturb beads: It's difficult during the first washing. At every washing steps : resuspend, put on rotating wheel for 5min and magnetic stand.

Wash 3 times with 0,5 mL of washing buffer I. After this washing, the beads appearance should be identical than in step 4a. If this is not the case, continue washing with buffer I. When the solution with beads appear “clear”:

Wash with 0,5 mL of washing buffer II.

Wash with 0,5 mL of washing buffer III.

Wash carefully twice with 0,5 mL TE8 (without incubation on rotating wheel).

On magnetic stand gently remove and discard all liquid taking care not to disturb beads.

Step 5 : Elution and Cross-Link Reversal :

For “IP” : Resuspend dried beads with 130 µL Elution Buffer.

For “INPUT” : Place tubes on magnetic stand and transfer the remaining supernatant (take all of it, included the foam) to a fresh tube. Add 1 µL of Proteinase K (at 20 mg/mL).

Incubate resuspended beads 5 to 6 hours at 65°C and vortex beads every hour.

After 5 or 6 h :

Vortex the tube and very low centrifugation (just for rescue all liquid).

Place “IP” tubes on magnetic stand for 5 minutes until the sample appears clear.

Gently transfer 130 µL of clear sample to new Low Binding Microcentrifuge Tube. Remove the tube of the magnetic stand and wash the beads with 20 uL Tris pH5.5 1M. On magnetic stand gently transfert the 20 µL of Tris to the 130 µL of clear sample (Vf = 150 µL).

For “INPUT” : Tubes are already ready for next step.

Step 6 : DNA recovery and cleanup : Choose version 1 or version 2

Version 1 : Cleanup with PCR Clean-up Columns (Macherey Nagel)

Cleanup the sample with the NucleoSpin® Gel and PCR Clean-up Columns (Macherey Nagel) and used the NTB (5 volumes of NTB for 1 of solution : see the kit procedure for samples with SDS) . Elute with 23 µL H2O millipore.

Version 2 : Cleanup with Chelex 100 Resin (Bio-Rad 1421253). Not tested yet.

Resuspend 0.2 g of Chelex with 1 mL H2O millipore : ready to use.

Add 100 uL of Chelex 20 % [Vortex manually the beads before each pipeting]. Incubate 30 minutes at 55°C (1000 rpm), gently spin to recover steam then incubate 10 minutes at 98 °C.

Centrifuge 1min30s at 13000 rpm. Take 135 uL of supernatant.

Control : If you want you can dose 1 µL of your sample with the Qubit 2.0 system (IP will probably be too low, INPUT around 3 ng/uL).

Use 1 µL for the HML and 1 µL for the HMR qPCR on Rotorgene.

 

Buffers, reagent list:

TBS 1X : 20mM Tris-HCl pH 7.5, 150mM NaCl (MCM stock)

Glycine 2,5M :

            for 50 mL : 9,383g (Long to dissolve, heating helps.)

Hepes 0.5M pH7.5 :

            For 100 mL : 11.9g Hepes.

            Adjust pH to 7.5 with K(OH)

Lysis Buffer :

Amount for 20mL stock

Final concentration

Sodium deoxycholate 5% w/v

400 µL

0.1% (w/v)

0.5M EDTA pH8

40 µL

1 mM

0.5M HEPES-KOH pH7.5

2 mL

50 mM

3M NaCl

932 µL

140 mM

Sterilize by autoclave add Triton X-100 10% and store at 4°C

Triton X-100 10%

2 mL

1% (w/v)

Prior to usage, add proteinase inhibitors (Sigma P8215 is ~6000 X)

Dynabeads prot-A (Life Technologies 10002D) (Stored at 4°C) See protocol for preparation (Step 4a).

Wash I : [5 mL]

Wash II : [5 mL]

Wash III : [5 mL]

0.1 mL TrisHCl 1M pH8

0.1 mL TrisHCl 1M pH8

0.05 mL TrisHCl 1M pH8

0.25 mL NaCl 3M

0.835 mL NaCl 3M

10 uL EDTA 0.5M pH8

20 uL EDTA 0.5M pH8

20 uL EDTA 0.5M pH8

0.5 mL déoxycholate 5%

0.5 mL TX-100 10%

0.5 mL TX-100 10%

0.5 mL Igepal 10%

0.05 mL SDS 10%

0.05 mL SDS 10%

0.625 mL LiCl 2M

TE8 : [TrisHCl 10mM, EDTA 1mM pH8.0][50 mL]

500 µL TrisHCl 1M pH8
100 µL EDTA 0.5M pH8

Tris 1M pH5.5

Elution buffer : [5 mL]                                                       Elution buffer Shearing : [2 mL]

150 µL Tris HCl 1M pH 8                                                    80 µL Tris HCl 1M pH 8
20 µL EDTA 0.5M                                                               80 µL EDTA 0.5M
500 µl SDS 10%                                                                   400 µl SDS 10%
87.5 µl proteinase K* (Roche 3115887001) 20 mg/ml         40 µl proteinase K (20 mg/ml) *

Qsp H2O 5 mL                                                                     Qsp H2O 2 mL

* add just before use

Phenol:Chloroform:Isoamyl Alcohol : [25:24:1] in light-tight bottle at 4°C.