ATCG

Single-Nucleosome Resolution Chromatin Immunoprecipitation from Yeast

Version 3.0

(Protocol Adapted for Magnetic beads by Filleton F).

Required Materials :

  • Culture media, TBS 1X, Buffer Z, NPS buffer, Mnase, EDTA, 10 % SDS, Proteinase K, Phénol , Chloroform, cold Ethanol 100% , TE, RNAse, IP buffer, washing buffer (I- II – III), TE8, Elution buffer, Tris pH5.5 1M, Glycogene NTB.

  • Glycine

  • Zymolyase

  • Beckman pot

  • Formaldehyde 37%

  • Dynabeads prot-A / prot-G (protocole not-tested with Dynabeads prot-G)

  • Antibodies of interest

  • Costar Low Binding Snap Cap Microcentrifuge Tubes

  • Magnetic Stand

  • Heat Block

  • Microcentrifuge

  • Rotating wheel

  • NucleoSpin® Gel and PCR Clean-up Columns (Macherey Nagel)

  • QIAGEN Minelute Reaction Cleanup Kit.

  • Vortex

  • Qubit 2,0 system

Step 1 : Preparation of cross-linked spheroplasts

(calculated for a generation time of 89 minutes)


At 11:30 a.m. Inoculate a starter culture of 50 mL SD+All medium at 30°C (in a 250mL flask)
At 5:00 p.m. Warm 200mL SD+All medium in a 1L flask at 30°C (plus at least 10mL in a tube)
At 5:45 p.m. Measure OD600 of the starter (= ODs).
At 6:00 p.m. Put 80/ODs μl of the starter into 5 mL of warm SD+All, mix and add all this mix to the 200 mL of warm medium. Grow overnight at 30°C.
Prepare glycine 2,5M (9,37g in 50ml distilled water), cold TBS 1x, zymolyase, and cool the centrifuge at 4°C
When OD600=1.4 (hopefully at about 10:00 a.m. the next morning), transfer 180mL of the culture to a Beckman pot and add 5ml of formaldehyde 37% ## FUME HOOD ## (final concentration 1%)
Incubate 15 min at room temperature on rotating rods
Add, under fume hood, 9 ml of glycine 2,5M (fresh prepared, role : quinch the cross linker).
Invert gently and incubate 5 min at room temperature on rotating rods
Transfert 200ml to four Falcon 50mL and centrifuge them for 5 min at 4000 rpm (~3000g) at 4°C on Jouan, rotor GR412.
Pour the supernatant in hazardous waste ## FUME HOOD ##  (containing formaldehyde)
Wash twice with 180 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).
Resuspend in 40ml cold TBS1x, transfer to Falcon 50ml tube, centrifuge at 3000g
Resuspend the yeast pellet in 39 ml Buffer Z (Pipet, Vortex).
Add 28 μl of β-MercaptoEthanol ## FUME HOOD ##  (14.3M, final conc 10mM)
Add 500 μl Zymolyase solution (10mg/ml = 200U/ml in Buffer Z, freshly prepared)
Incubate at 30°C, shaking overlunch.

Step 2 : Micrococcal nuclease digestion

Prepare NP-S buffer by adding protease inhibitor cocktail (Sigma P8215 is ~6000 X)

Check lysis under microscope (compare aspect of 6 μl cells with or without 6 μl 0.1% SDS).
Spin 3000g at 4°C for 10 min.
Resuspend the pellet in 1200 μl of NP-S Buffer + ProteaseInhibitors, split into two halves that can be processed separately for all the following steps (to test two different antibodies for example) and put on ice. SnapFreeze in liquid N2 and store the extra aliquots at -80°C.
To a 600 μl sample of spheroplasts, add ~12µl (depends on the strain) of Microccal Nuclease. Incubate at 37°C for 1h30. Put on ice and add 12.2 μl of EDTA 0.5M (final conc  = 10mM) to stop reaction. If necessary: withdraw 40 μl of sample and SnapFreeze the rest in liquid N2 and store at -80°C. Check digestion on 2% agarose gel using the 40 μl you drew (see next step).

Step 3 : Micrococcal Nuclease Digestion Quality Control

You can verify efficient separation of nucleosomes by the following:
1. mix 20µl extract +1µl 10% SDS + 0.5µl proteinase K
2. incubate 3 hrs at 65°C
3. phenol/chloroform extract the samples
4. chloroform extract the samples
5. Precipitate DNA : add 2volumes of cold Ethanol and glycogen to 10 µg/ml final concentration.
6. resuspend DNA in 20 µl TE
7. treat with 40 µg/ml RNAse for 1hr at 37 degrees C
8. run half of each sample on a 2% agarose gel with an appropriate DNA sizing ladder (DNA from single nucleosomes should run at about 150bp)

Step 4a:  Dynabeads pA preparation

Beads are stored at 4 °C.

[ Predict 30 µl of beads pA and X µl of antibody (depends on the antibody manual).  For 6 IPs = 190 µl of pA beads and 6*X µl of antibody. Vortex manualy the beads stock solution before each pipeting.]

At room temperature with sterile gloves :

Transfer the required volume of beads in a 1.7 ml Costar tube (Low Binding Snap Cap Microcentrifuge Tubes). 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 plate from magnetic stand and resuspend beads with 500 µl of IP buffer. Repeat the previous step 2 other times to wash the bead (total of 3 washings).

Resuspend the beads in 1 volume (~30 µl) of IP buffer (for 6 IP = 190 µl ).

Add in the tube the corresponding quantity of antibody (X µl * nb of IP). [For step 5, no antidoby for the MNase-seq sample].

Incubate beads on rotating wheel at room temperature for 20 minutes.

Step 4B:  Immunoprecipitation

In a new Costar tube, transfer 550 µl of the remaining supernatant obtain by MNase digestion ( “IP” tube). Store the rest of supernatant at -80°C (INPUT control fraction).

Add 30 µl of previously prepare Dynabeads pA preparation ( Beads + antibody). [Vortex manually the beads before each pipeting.]

At 4 °C , incubate the final “IP” overnight on a rotating wheel.

At morning, put the tube 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 3a. 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 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.

Resuspend dried beads with 130 µl Elution Buffer.

Incubate resuspended beads 6h at 65 °C and vortex beads every 1h30-2h.

After 6h:

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

Place tube on magnetic stand for 5 minutes until the sample appears clear.

Gently transfer 130 µl of clear sample to new Costar tube.

Remove the tube of the magnetic stand and wash the beads with 20 μl Tris pH5.5 1M. On magnetic stand gently transfert the 20 μl of Tris to the 130 µl of clear sample (Vf= 150 μl)

 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.

Dose 1 µl of your sample with the Qubit 2.0 system.

Control : Use 1 µl for the HML ans 1 µl for the HMR qPCR on rotor gene

Step 5:  MNase-seq sample

Prepare your Dynabeads without antibody (see step 4a)

In a Costar tube, put 550 µl of the remaining supernatant obtain by MNase digestion and add 30 µl of previously prepare Dynabeads pA preparation ( Beads alone).

Incubate 1hour on a rotating wheel.

Put the tube on the magnetic stand until the magnetic beads appears magnetized.

Transfer the remaining supernatant (take all of it, including the foam) to a fresh tube.

Add 200µl of 4X Buffer L + protease inhibitors, mix by inversions.

1. mix 200µl of extract (other ul are stock at -80°C) +10µl 10% SDS + 5µl proteinase K
2. incubate 3 hrs at 65°C
3. phenol/chloroform extract the samples
4. chloroform extract the samples
5. Precipitate DNA : add 2volumes of cold Ethanol and glycogen to 100 µg/ml final concentration.
6. Resuspend DNA in 200 µl TE
7. Treat with 400 µg/ml RNAse for 1hr at 37 degrees C.

8. Cleanup the sample with the QIAGEN Minelute Reaction Cleanup Kit. Elute with 20-40µl H2O millipore (depending of your Mnase sample concentration).

9. Dose with the Qubit 2.0 system

Buffers, reagent list:

 TBS 1X :

20mM Tris-HCl pH 7.5, 150mM NaCl

Glycine 2.5M :
for 500 mL : 93,83g
Long to dissolve, heating helps.

Buffer Z :
Amount for 1L stock Final concentration
Sorbitol 182 g 1 M
Tris-Cl pH7.4 (1M)50 mL 50 mM
Autoclave, store at 4°C

Zymolyase :

ref 120491 from SEIKAGAKU distributed by MPbiomedicals.

Micrococcal Nuclease:

ref LS004797 (15000 units) from Worthington Biochemicals, distributed by Serlabo. Dissolve in 1.67mL of [500µl glycerol (MB grade) + 1167µl H2O millipore], and store in 70µl aliquots at -20°C.

NP-S Buffer :

Amount for 200mL stock Final concentration
Spermidine (MW = 145.25) 14,4 mg 0.5 mM
NP-40 (100%) 150 μL 0.075%
Tris-Cl pH7.4 (1M) 2 mL 10mM
NaCl (5M) 2 mL 50mM
MgCl2 (1M) 1 mL 5mM
CaCl2 (1M)200 μL 1mM
β-MercaptoEthanol (98%=14,3M) 14 μL 1mM
Store at 4°C.
Prior to usage, add proteinase inhibitors (Sigma P8215 is ~6000 X)

4X Buffer L:

Amount for 1L stock Final concentration
Sodium deoxycholate 4 g 0.4% (W/V)
0.5M EDTA 8 mL 4 mM
1M HEPES pH7.5 200 mL 200 mM
5M NaCl 82 mL 410 mM
Sterilize by autoclaving, add 40 mL of Triton-X100 (final conc 4% V/V), Store at 4°C
Prior to usage, add proteinase inhibitors (Sigma P8215 is ~6000 X)

 Elution buffer [5 ml] :

150 uL Tris HCl 1M pH 8
20 uL EDTA 0.5M
500 ul SDS 10%
175 ul proteinase K Roche 10 mg/ml

1ml TE, 0.5% SDS, Prot K :

880µL TE pH8
50 ul SDS 10%
70 ul proteinase K Roche 10 mg/ml

 IP buffer [10 ml] :

Tris/HCl 1M pH 8 200 µl
NaCl 3M 500 µl
EDTA 0.5M pH8 40 µl
Triton X-100 10% 1 ml
H2Oqsp
store at RT

 TE8 :
10 mM Tris HCl pH 8, 1mM EDTA pH 8

 TE [10 ml] :
Tris-HCl 1M, pH8 100 µl
EDTA 0.5M, pH8 20 µl

Elution buffer:
TE pH 8, SDS 1%, 0.35 mg/ml proteinase K

Wash I [5 ml]:
0.1 ml Tris/HCl 1M pH8
0.25 ml NaCl 3M
20 µl EDTA 0.5M pH8
0.5 ml TX-100 10%
0.05 ml SDS 10%

Wash II [5 ml]:
0.1 ml Tris/HCl 1M pH8
0.835 ml NaCl 3M
20 µl EDTA 0.5M pH8
0.5 ml TX-100 10%
0.05 ml SDS 10%

Wash III [5 ml]:
0.05 ml Tris/HCl 1M pH8
10 µl EDTA 0.5M pH8
0.5 ml déoxycholate 5%
0.5 ml Igepal 10%
0.625 ml LiCl 2M

Protéinase K: Roche 03115879001
Dynabeads prot-A: life technologies 10002D [Dynabeads prot-G: life technologies 10004D]
Costar tubes, Corning® 1.7mL Low Binding Snap Cap Microcentrifuge Tubes (costar 3207)
Macherey Nagel 740609 (magasin: MN06)
NTB buffer: Macherey Nagel 740595