Chromatin immunoprecipitation (ChIP) assay

ChIP protocol

Reagents needed:

1.)   Fix buffer: 20X stock-1litre

100ml               1M Hepes pH 8.0          (final conc. 100mM)

2ml                  0.5M EGTA pH 8.0       (final conc. 1mM)

40ml                5M NaCl                      (final conc. 200mM)      

Milli Q water                Final volume 1 litre

2.)   Prepare a mix of Fix Buffer + Formaldehyde:

50ml                20X Fix buffer stock     (final conc. 10X)

30ml                Formaldehyde 37%       (final conc. 11%)

20ml                MilliQ water                 Final volume 100ml

3.)   SDS Lysis Buffer:

50ml                 1M Tris pH 8.1             (final conc.50mM)

10ml                0.5M EDTA pH 8.0       (final conc. 5mM)

20ml                5M NaCl                      (final conc. 100mM)

50ml                10%SDS                                   (final 0.5%v/v)

Total Volume 1litre with MilliQ water

4.)   Triton Dilution Buffer

      25ml                 1M Tris-Cl, pH 8.6 (100mM Final)

        5.0ml              5M NaCl (100mM Final)

               2.5ml              0.5M EDTA, pH 8.0 (5mM Final)

             500ml                10% NaN₃ (0.2% Final)

              62.5ml             20% Triton X-100 (5.0% Final)

              Add dH₂0 to 250ml

1)     For adherent cells add Fix-Formaldehyde buffer to the media directly in the dishes, so that it is diluted 1:10. (e.g. if cells are in 20ml medium then add 2.2 ml of fix-formaldehyde buffer so finally it is 1%formaldehyde and 1X buffer). 

For suspension cells, pellet suspension cells, and adds appropriate volume (20ml) of 37°C pre-heated 10%FBS MEDIA followed by adding Fix-Formaldehyde buffer (Nr.2)  in 1:10 dilution.

2)     Fix at room temperature for 10 min.

3)     Stop fixation by adding glycine to final concentration of 0.125 M and incubate for 5 min (Glycine stock is 2M).  Swirl plates or invert tubes in between.

4)     Wash 2x with 1X PBS at room temperature.

5)     Aspirate PBS completely from plate and add SDS Buffer containing protease inhibitors (Aprotin+Leupeptin+PMSF). 1ml SDS lysis buffer per 15cm dish. (Normally, collect cells up to 15 x 15 cm dishes in 10 ml of SDS Buffer). Scrape cells and collect in the tube. Snap freeze the tubes after collecting all plates. For suspension cells, 5-10X10^6 cells per ml of SDS lysis buffer, so if having 100X10^6 cells then add 10ml of SDS lysis buffer and divide it into 2- 15ml falcon tubes.

FIXED CELLS CAN BE FROZEN AT -80°C in SDS Buffer

6)     Thaw cells from freezer in a water bath at RT. Be sure that all the SDS went back in solution.

7)     Pellet cells by spinning in tabletop centrifuge for 6 min at 1200 rpm.

8)     Resuspend cells in appropriate volume of ice-cold IP Buffer for sonication (IP buffer =1 volume SDS Buffer : 0.5 volume Triton Dilution Buffer). The volume depends by the kind of sonicator you are using and from how many cells you have fixed. 

9)     Sonicate samples to an average length of 500-1,000 bp (e.g. pulse for 20-30 sec at power setting 30% for 5-6 times on ice with 2-3 min. rest in between with the large tip of a Branson Sonifier (5x15cm in 5ml IP buffer), Alternatively if using Bioruptor, the sonication in 1.3ml (3x15cm in 1.3ml IP buffer) for 10-12 min with a 30sec/30sec cycle). In general sonication conditions need to be optimized between machines and between different cell lines.

10)  Centrifuge sonicated chromatin at 20000g for 30 min.

11)   Transfer supernatant to a new tube.

12)  Take a 25-50µl aliquot for sonication control. Add final 1%SDS, 0.1M NaHCO3 and de-crosslink min 3h

13)  Purify DNA with QIAGEN (or similar) PCR purification columns.

14)  Run purified DNA on a 1% Agarose gel

15)  If desire sonication is achieved proceed with the next step. Otherwise proceed with further sonication.

16)  Quantify DNA conc. (from step 13) using nanodrop. 

17)  Backcalculate the chromatin conc. and use 20ug chromatin per IP for transcription factors and 5ug-10ug for histone modification.

18)  Dilute sample with IP buffer to a desired concentration. Consider performing each single IP in 1ml in loDNA bind tubes (Eppendorf).

19)  Remove 10ml of lysate to be used as total control (=1%).  Keep at 4 ⁰C.

20)  Aliquot chromatin in 1ml fractions

21)  Add primary antibody and incubate overnight at 4⁰C rotating.  

22)  Wash proteinA+proteinG beads in IP wash buffer. 

23)  Using a wide-bore pipet tip add 30-40 ml of Beads. Incubate on a rotating wheel 2-4 hr at 4⁰C.

24)  Microcentrifuge beads 1 min at 1800g at 4°C.  

25)  Wash beads for 3 times with 1.5 ml of 150mM Wash Buffer

26)  Wash beads for 1 time with 1.5 ml of 500mM Wash Buffer 

27)  Add 120 ml of 1% SDS, 0.1M NaHCO₃ and incubate min 3h at 65⁰C (shaking) to elute complexes from beads. Remember to include the input by adding 120 µl of 1% SDS, 0.1M NaHCO₃ to 10µl of input chromatin.

28) For reverse crosslinking, add 1 μl RNaseA (1mg/ml stock) and 18 μl 5M NaCl (final conc. 0.3M). Add 1 μl RNaseA and 10.8 μl 5M NaCl to input samples. Incubate at 67^oC overnight.

29)  Add 600µl of PB buffer (Qiagen PCR purification kit) and proceed with purification without worrying too much about the beads (they stay in the column).

30)  Elute 2 times in 100µl of H₂O or EB buffer and use 1-2µl per q-real time PCR

5.)   150mM Wash Buffer

1% Triton X-100

0.1% SDS

150mM NaCl

2mM EDTA, pH 8.0

20mM Tris-HCl, pH 8.0

6.)   500mM Wash Buffer

1% Triton X-100

0.1% SDS

500mM NaCl

2mM EDTA, pH 8.0

20mM Tris-HCl, pH 8.0

Protease Inhibitors. 

5mg/ml PMSF in EtOH, use at 1:100 (working conc. 50μg/ml) 

1mg/ml leupeptin in water, use at 1:1000 (working conc 1μg/ml) Store at -20 C 

1M Sodium butyrate, use at 1:100 (Working conc. on 10mM) Store at 4 C 

2M Glycine stock (prepare in water)

Glycogen [Invitrogen Cat. No. 10814-010 (100μl)]  1mg/ml stock 

RNaseA [Sigma-Aldrich Cat. No. R6513-10MG] 1mg/ml stock 

Other Reagents required. 

TE pH8.0 

Formaldehyde 

Protein A/G sepharose [Protein G Agarose –Roche-diagnostics Cat. No. 11719416001 (2ml)] 

5M NaCl 

100% ethanol 

70% ethanol 

Proteinase K (20mg/ml) 

3M NaAc 

Autoclaved water