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Chromatin Immunoprecipitation (ChIP) Protocol using Protein A beads (Dorsky Lab)

Shan-Fu’s adaptation from Kazuyuki’s protocol (Grunwald Lab) (8/24/05)

Modified by Junji Lin (06/29/2010)

<Prior to Day1>

¨    Coating protein A beads

Take 50% protein A beads slurry (about 50ul for each sample) (Invitrogen #15918-014)

Spin 1.5k rpm, 30 sec at RT

Remove the sup. with loading tip

Wash three times with 1 mL IP dilution buffer

Resuspend beads in 1mL pre-blocking buffer (10 mg/mL calf thymus DNA 100 uL, 20 mg/mL BSA 50 uL, and IP dilution buffer 850 uL) and incubate at 4oC, O/N

Spin 1.5k rpm, 30 sec at RT

Remove the sup.

Wash twice with 1 mL IP dilution buffer

Add IP dilution buffer back to original volume

(Comments: This protocol is not for ChIP-Seq since other types of carrier DNA (such as calf thymus DNA) were used for blocking. Washing and blocking protein A beads are necessary for eliminating non-specific beads’ binding.)

<Day 1>

¨    Cross-linking of protein and DNA

Dechorinate 100 embryos in 1xER, then remove the supernatant (sup.)

Add 1% formaldehyde in 1xPBS in 1.5 mL tube

Rotate 15 min at RT, remove sup.

Rinse with 1 mL 0.125 M glycine

Add 0.125 M glycine to soak embryos, rotate 10 min at RT

Rinse embryos with 1 mL cold 1xPBS twice

(Comments: The amount of embryos needed for ChIP need to be optimized by specific sample. It also depends on your antibody specificity. As far as I know, I can see difference between negative control and sample with only 10 embryos using dynabeads. But I haven’t check it with protein A beads.

                   Cross-linking condition are based on100 embryos at 36hpf. Time will vary if using younger or older embryos; 2.2% of formaldehyde can be used if necessary. Insufficient cross-linking will lose true binding signals and over cross-linking will bring non-specific binding. )

¨    Cell lysis and chromatin DNA extraction

Add 600 uL Cell lysis buffer (for 100 embryos), on ice 10~20 min

Pipet up and down every 5-10min until no clear tissue can be visible (bone structure might be still there, but most of the tissue should be broken and dissolved)

Cfg: 3.5k rpm, 5 min at 4 oC, remove the sup.

Rinse once more with 600 uL Cell lysis buffer and spin down, remove the sup.

Re-suspend the nuclear pellet (white) in 200 uL nuclei lysis buffer

Pipet up and down to disrupt clumps

On ice 10~20 min (lay the tube on ice to avoid precipitation of SDS in the nuclei lysis buffer)

Add 400 uL IP dilution buffer + proteinase inhibitors

Freeze at -70 or -80 oC

¨    Sonication

Thaw the sample and add 100 mg glass beads (Sigma, acid washed, 212-300 microns, Sigma #G-1277)

Sonication machine (Branson Sonifer 250 with microtip)

Set up: constant duty cycle, output on 2, value 20

6 pulses of 20 sec each with 3 min interval on ice

Take 10 uL for agarose gel electrophoresis to check the size of fragmented DNA after decross-linking

(Comments: Over sonication will lead to DNA degradation, Insufficient sonication will lead to non-specific binding. Average fragment size should be between 100bp-600bp. Avoid introducing bubbles to the sample or generating too much heat since it will lead to protein degradation. Keep on ice during sonication and leave the sample on ice for enough time to cool down before next round of sonication.)

Cfg: 14k rpm, 15 min at 4oC

Take the sup. Add IP dilution buffer to 600 uL per tube.

Divide equal proportionally for ChIP and total input control. (For example: total amount after sonication is 600ul, using 500ul for ChIP assay and 100ul for input control for each sample) Store input sample at -20 oC.

¨    Antibody binding

Pre-clear:

Add 40 uL coated protein A beads (in pre-Day1) to the ChIP sample

Rotate at 4oC, 15 min

Antibody binding:

Cfg: 5k rpm, 10 min, 4oC

Take the sup. (Do not contaminate with any beads) and divide it equally as antibody and no-antibody control.

Add antibody 5ug to the sample (Keep the no-antibody control sample at 4 oC without adding any antibody) rotate O/N at 4oC

<Day 2>

¨    Protein A beads binding, washing, elution, and decross-linking

Add 40 uL blocked 50% protein A beads slurry (also to no-antibody control tube)

Incubate on a rotating wheel/platform at RT, >15 min

Cfg: 1.5k rpm, 4 min at RT,

Remove sup. (Beads from no-antibody tube could serve as “no Ab” negative control after washing and elution)

Wash the beads twice with 1 mL 1x dialysis buffer

           Add 1 mL buffer

           Rotate 15 min at RT

           Cfg: 1.5k rpm, 4 min at RT

           Remove the sup. as much as possible with loading tip

Wash the beads twice with 1 mL IP wash buffer (same as above)

(Comment: Washing step could be held at 4 oC with longer time; If non-specific binding still occur, wash 3 times for each buffer followed by 1~3 times TE wash)

¨    Elution and decross-linking

Add 150 uL elution buffer to the beads

Rotate 15 min at RT

Cfg: 14k rpm, 4 min at RT

Transfer the sup. to a new tube

Repeat elution steps again and combine both elution (300 uL).

Add 30 uL 3 M NaCl and 1 uL 10 mg/mL RNaseA (For input sample, add right amount of NaCl and RNaseA)

Incubate 65 oC, 4-5 hr or O/N (input control samples also need to be decross-linking at the time)

Add 2 uL 5 mg/mL glycogen and 2.5V absolute EtOH, -80oC O/N

<Day 3>

¨    Proteinase K treatment

Cfg: 14k rpm, 4oC, 20 min

Discard sup.

Dissolve pellet in TE, and mix with            5x PK buffer       PK(10 mg/mL)

  For ChIP sample 100 uL                   25 uL             2 uL

  For Input sample 200 uL                   50 uL             2 uL

45oC, 1-2 hr

Add TE to 300 uL

Add 300 uL phenol/CHCl3, vortex, 14k rpm, 5 min at RT, take the sup.

Add 300 uL CHCl3, vortex, 14k rpm, 5 min at RT, take the sup.

Add 3 M NaCl 54 uL, 5 mg/mL glycogen 2 uL, 2.5V absolute EtOH

-80oC, O/N  

<Day4>

¨    Precipitation and PCR

14k rpm, 20 min at 4oC

Dissolve pellet in 10-30 uL TE or ddH2O

Further DNA purification can use QIAquick PCR purification Kit if necessary. But it will also lose quite a bit amount of DNA.

DNA can be stored at -20 oC

qPCR with ChIP sample, no-Ab control and total input.

¨    Recipes for solution and material information

1 M glycine (FW = 75.07)

         3.75 g / 50 mL dH2O

         filtrate and store at RT

100x ER

         6 g instant ocean / 1 L dH2O

1% formaldehyde in 1x PBS (freshly prepared)

         Total               25ml    

         37% formaldehyde   676 uL    

         1x PBS            24.324ul

0.125 M glycine in 1x PBS (freshly prepared)

                                          10 mL        30 mL

         1 M glycine                               1.25 mL     3.75 mL    

         1x PBS                                8.75 mL    26.25 mL

Proteinase inhibitors

        (final conc.)                       (per 1 mL solution/buffer)

         100 uM PMSF                            1 uL of 100 mM

         100 ug/mL benzamideine                       10 uL of 10 mg/mL

          5 ug/mL leupeptin/pepstatin                           1 uL of 5 mg/mL 

Cell lysis buffer (at 4oC)                         (40 mL)

         10 mM Tris-Cl (pH 8,1)             400 uL of 1 M   (Using Tris Base)

         10 mM NaCl                              133 uL of 3 M

          0.5% NP-40                          2 mL of 10%

          proteinase inhibitors (freshly add)

          dH2O                                                              37.47 mL

                                            

Nuclei lysis buffer                                          (20 mL)

 50 mM Tris-Cl (pH 8.1)                                                  1 mL of 1 M  

10 mM EDTA                                                 400 uL of 0.5 M

 1% SDS (Sodium Dodecyl Sulfate)                                                 2 mL of 10%

 proteinase inhibitors (freshly add)

 dH2O                                                     16.6 mL

IP dilution buffer (at 4oC)                                   (40 mL)

           16.7 mM Tris-Cl (pH 8.1)                      668 uL of 1 M 

           167 mM NaCl                              2227 uL of 3 M

           1.2 mM EDTA                                96 uL of 0.5 M

           1.1% Triton X-100                             2.2 mL of 20%

           0.01% SDS                                  40 uL of 10%

           proteinase inhibitors (freshly add) 

           dH2O                                                                       34.77 mL                                                        

Pre-Blocking Buffer: 1 mg/mL calf thymus DNA and 1 mg/mL BSA in IP dilution buffer (1 mL)                                                                          

            10 mg/mL BSA                                  100 uL

            10 mg/mL denatured calf thymus DNA            100 uL

            IP dilution buffer                                  800 uL

500 mM NaHCO3 (filtrate, store at RT)

             NaHCO3 (FW = 84.01)                                       4.2 g

             H2O                                       up to 100 mL

1x dialysis buffer (store at 4oC)                           (100 mL)

    50 mM Tris-Cl (pH 8.1)                                5 mL of 1 M

    2 mM EDTA                                      400 uL of 0.5 M

    0.2% sarkosyl (omit for monoclonal Ab)                     1 mL of 20%

    The other name is N-lauroylsarcosin solution (SIGMA L7414-10ML)                                                                    

dH2O                                                                                      92.6 mL

IP wash buffer (stored at 4oC) (100 mL)                                                    

100 mM Tris-Cl (pH 9.0) (pH 8.0 for monoclonal Ab)                   10 mL of 1 M

500 mM LiCl                                                                                      10 mL of 5 M                                                            

1% NP-40                                                                                              10 mL of 10%                                                                       

1% deoxycholic acid (Sodium deoxycholate Sigma D6750-25G)      5 mL of 20%

dH2O                                                                                                       65 mL                                                                                                    

Elution buffer                                                    (40 mL)

           50 mM NaHCO3                                       4 mL of 500 mM

           1% SDS                                       4 mL of 10%

           dH2O                               32 mL of dH2O  

   

5x PK buffer

          50 mM Tris-Cl (pH 7.5)                       5 mL of 1 M

          25 mM EDTA                       5 mL of 0.5 M

          1.25% SDS                        12.5 mL of 10%

          dH2O                                                       77.5 mL

                                        

Glass beads, acid-washed, Sigma G1277-100G

10 mg/mL proteinase K

10 mg/mL glycogen

3 M NaCl

Phenol (saturated with TE)

CHCl3 (+1/25 vol. Iso-amyl alcohol)

EtOH (-20oC)