ChIP using plant samples

实验概要

The  immunoprecipitation (IP) of cross-linked chromatin with antibodies  specific for certain histone modifications (chromatin  immunoprecipitation, ChIP), followed by PCR to detect a potential  enrichment or depletion of a DNA sequence of interest within IP  fractions, constitutes an elegant and direct method to query specific  chromatin states of individual genes.

实验原理

Eukaryotic  chromatin is a complex of DNA and associated histone proteins that are  involved in the higher order packaging of DNA into chromosomes. The  chromatin state of a given DNA sequence influences transcriptional  activity and replication timing and is regulated by potentially  reversible covalent modifications of DNA and histones. Histone  modifications at conserved lysine and arginine residues within the  flexible N-terminal tails, such as phosphorylation, acetylation and  methylation, specify a code that serves as an interaction platform with  specific domains of chromatin-associated proteins. The  immunoprecipitation (IP) of cross-linked chromatin with antibodies  specific for certain histone modifications (chromatin  immunoprecipitation, ChIP), followed by PCR to detect a potential  enrichment or depletion of a DNA sequence of interest within IP  fractions, constitutes an elegant and direct method to query specific  chromatin states of individual genes and is already routinely used in  many labs. In contrast to animal cells, however, plant cells have a  rigid cell wall which poses limitations to the simple utilization of  protocols established for animals. In this protocol, the method  described in used to study histone modifications in the plant model  organism Arabidopsis thaliana. This protocol is an adapted version of the original procedure published by Lawrence and co-workers.

This  protocol describes how chromatin is prepared from Arabidopsis, which  can subsequently be used for chromatin immunoprecipitation (ChIP). The  exact chromatin concentration should be determined before starting the  X-ChIP assay.  

主要试剂

Extraction buffer 1

0.4 M Sucrose

10 mM Tris-HCl, pH 8.0

10 mM MgCl2

5 mM β-mercaptoethanol

Protease inhibitors

Extraction buffer 2

0.25 M Sucrose

10 mM Tris-HCl, pH 8.0

10 mM MgCl2

1% w/v Triton X-100

5 mM β-mercaptoethanol

Protease inhibitors

Extraction buffer 3

1.7 M Sucrose

10 mM Tris-HCl, pH 8.0

2 mM MgCl2

0.15% w/v Triton X-100

5 mM β-mercaptoethanol

Protease inhibitors

Nuclei lysis buffer

50 mM Tris-HCl, pH 8.0

10 mM EDTA

1% w/v SDS

Protease inhibitors

Protease inhibitor

100 mM PMSF

One tablet per 10ml of solution of complete mini protease inhibitor cocktail tablets (Roche®)

FA Lysis Buffer

50 mM HEPES-KOH pH7.5

140 mM NaCl

1 mM EDTA pH8

1% Triton X-100

0.1% Sodium Deoxycholate

0.1% SDS

Protease Inhibitors (add fresh each time)

RIPA Buffer

50 mM Tris-HCl pH8

150 mM NaCl

2 mM EDTA pH8 1% NP-40

0.5% Sodium Deoxycholate

0.1% SDS

Protease Inhibitors (add fresh each time)

Wash Buffer

0.1% SDS

1% Triton X-100

2 mM EDTA pH8

150 mM NaCl

20 mM Tris-HCl pH8

Final Wash Buffer

0.1% SDS

1% Triton X-100

2 mM EDTA pH8

500 mM NaCl

20 mM Tris-HCl pH8

Elution Buffer

1% SDS

100mM NaHCO3

实验材料

Arabidopsis  seeds are stratified for 48 hours in 0.1% Phytablend w/v at 4°C and  then sown onto soil. 1.5 g of whole, three to four week-old seedlings,  are used per chromatin preparation. It is imperative to avoid  contamination with soil as much as possible during harvest.

实验步骤

1.        Chromatin cross-linking

1)      Harvest 1.5 g seedlings and place them into a 50 ml tube.

2)      Rinse seedlings twice with 40 ml double distilled water. Remove as much water as possible after second wash.

3)      Add  37 ml 1% w/v formaldehyde solution. Gently submerge seedlings at the  bottom of the tube by stuffing the tube with nylon mesh. Screw on cap  and poke cap with needle holes. Put in exsiccator and draw vacuum for  ten minutes.

4)      Release vacuum slowly and shake exsiccator slightly to remove air bubbles. Seedlings should appear translucent.

5)      Add 2.5 ml 2 M glycine to quench cross-linking. Draw vacuum for five minutes.

6)      Again, release vacuum slowly and shake exsiccator slightly to remove air bubbles.

7)      Remove  nylon mesh, decant supernatant and wash seedlings twice with 40 ml of  double distilled water. After second wash, remove as much water as  possible and put seedlings between two layers of kitchen paper. Roll up  paper layers carefully to remove as much liquid as possible.

At this step, plant material can be snap-frozen in liquid nitrogen and stored at -80°C

2.        Chromatin preparation

1)      Pre-cool  mortar with liquid nitrogen. Add two small spoons of white quartz sand  (Sigma Cat.: 27,473-9) and plant material. Grind plant material to a  fine powder.

2)      Use  cooled spoon to add powder to 30 ml of extraction buffer 1 stored on  ice. Vortex to mix and keep at 4°C until solution is homogenous.

3)      Incubate for 30 min at 4°C with gentle agitation.

4)      Filter extract through into a new, ice-cold 50 ml conical tube. Press to recover extract from solid material.

5)      Repeat step four.

6)      Centrifuge extract at 4000 rpm for 20 minutes at 4°C.

7)      Gently  pour off supernatant and resuspend pellet in 1 ml of extraction buffer 2  by pipetting up and down. Transfer solution to Eppendorf tube.

8)      Spin in cooled benchtop centrifuge at 13000 rpm for ten minutes.

9)      Remove supernatant and resuspend pellet in 300 µl of extraction buffer 2 by pipetting up and down.

10)  Add 300 µl of extraction buffer 3 to fresh Eppendorf tube. Use pipette to carefully layer solution from step nine onto it.

11)  Spin  in cooled benchtop centrifuge at 13000 rpm for one hour. In meantime,  prepare 10 ml nuclei lysis buffer. Put buffers in coldroom.

12)  Remove  supernatant and resuspend pellet in 300 to 500 µl of cold nuclei lysis  buffer. Resuspend by pipetting up and down and by vortexing. Keep  solution cold between vortexing. Incubate for 20 minutes on ice.

13)  Remove 10 µl to run on an agarose gel.

14)  Sonicate for ten minutes at 4°C.

15)  Spin in cooled benchtop centrifuge at 13000 rpm for ten minutes. Add supernatant to new Eppendorf tube.

16)  Repeat step 14. Remove 10 µl to run on an agarose gel

17)  Separate  aliquots from steps 12 and 15 on 1.5% w/v agarose gel. In the sonicated  samples, DNA should be shifted and more intense compared to untreated  samples and range between 200-2000 bp, centering around 500 bp.

3.        Cross-linking and cell harvesting

Formaldehyde  is used to cross-link the proteins to the DNA. Cross-linking is a time  dependent procedure and optimization will be required. We would suggest  cross-linking the samples for 2 - 30 min. Excessive cross-linking  reduces antigen accessibility and sonication efficiency. Epitopes may  also be masked. Glycine is added to quench the formaldehyde and  terminates the cross-linking reaction.

1)      Start with two confluent 150 cm²  dishes (1x107- 5x107 cells per dish). Cross-link proteins to DNA by  adding formaldehyde drop-wise directly to the media to a final  concentration of 0.75% and rotate gently at room temperature (RT) for 10  min.

2)      Add glycine to a final concentration of 125 mM to the media and incubate with shaking for 5 min at RT.

3)      Rinse cells two times with 10 ml cold PBS.

4)      Scrape cells into 5 ml cold PBS and transfer into 50 ml tube.

5)      Add 3 ml PBS to dishes and transfer the remainder of the cells to the 50 ml tube.

6)      Centrifuge for 5 min, 1,000 g.

7)      Carefully aspirate off supernatant and resuspend pellet in FA Lysis Buffer (750 μl per 1x107 cells).

When  using suspension cells, start with 1x107- 5x107 cells and treat with  both 0.75% formaldehyde and glycine as described above (Section 1).  Pellet cells by centrifugation (5 mins,1,000 g). Wash 3 times with cold  PBS and resuspend pellet in FA Lysis Buffer (750 μl per 1x107 cells).  Proceed to Step 2.1.

4.        Sonication

1)      Sonicate  lysate to shear DNA to an average fragment size of 500 - 1000 bp. This  will need optimizing as different cell lines require different  sonication times.

The  cross-linked lysate should be sonicated over a time-course to identify  optimal conditions. Samples should be removed over the time-course and  DNA isolated as described in Section 3. The fragment size should be  analyzed on a 1.5% agarose gel as demonstrated in Figure 1.

2)      After  sonication, pellet cell debris by centrifugation 30 sec, 4°C, 8,000 g.  Transfer supernatant to a new tube. This chromatin preparation will be  used for the immunoprecipitation (IP) in Step 4.

3)      Remove  50 μl of each sonicated sample, this sample is the INPUT. This is used  to quantify the DNA concentration (see Step 3) and as a control in the  PCR.

The  sonicated chromatin can be snap frozen in liquid nitrogen and stored at  -70°C for up to 2 months. Avoid multiple freeze-thawing.

5.        Determination of DNA concentration

1)      The  INPUT samples are used to calculate the DNA concentration for  subsequent IPs. The DNA is purified using either a PCR purification kit  (add 70 μl of Elution Buffer and proceed to Step 3.2a) or  phenol:chloroform (add 350 μl of Elution Buffer and proceed to Step  3.2b).

2)      a.  Add 2 μl RNase A (0.5 mg/ml). Heat with shaking at 65°C for 4-5 hr (or  overnight) to reverse the cross-links. DNA is purified using a PCR  purification kit according to the manufacturer’s instructions. The  samples can be frozen and stored at -20°C.

Samples  are treated with RNase A as high levels of RNA will interfere with DNA  purification when using the PCR purification kit. Yields can be severely  reduced as the columns become saturated.

3)      b.  Add 5 ul proteinase K (20 mg/ml). Heat with shaking at 65°C for 4-5 hr  (or overnight) to reverse the cross-links. The DNA is phenol:chloroform  extracted and ethanol precipitated in the presence of 10 μl glycogen (5  mg/ml). Resuspend in 100 μl H₂O. The samples can be frozen and stored at -20°C.

Samples  are treated with proteinase K, which cleaves peptide bonds adjacent to  the carboxylic group of aliphatic and aromatic amino acids. Cross-links  between proteins and DNA are disrupted which aids DNA purification.

4)      To  determine the DNA concentration, transfer 5 μl of the purified DNA into  a tube containing 995 μl TE to give a 200-fold dilution and read the  OD260. The concentration of DNA in μg/ml is OD260 x 10,000. This is used  to calculate the DNA concentration of the chromatin preparation.

6.        Immunoprecipitation

1)      Use  the chromatin preparation from Step 2.2, an equivalent amount of  approximately 25 μg of DNA per IP is recommended. Dilute each sample  1:10 with RIPA Buffer. You will need one sample for the beads-only  control.

2)      Add  primary antibody to all samples except the beads-only control. The  amount of antibody to be added should be determined empirically; 1-10 μg  of antibody per 25 μg of DNA often works well.

3)      Add  20 μl of protein A/G beads (pre-adsorbed with sonicated single stranded  herring sperm DNA and BSA, see step 4.3a) to all samples and IP  overnight with rotation at 4°C.

4)      a.  Preparation of protein A/G beads with single stranded herring sperm  DNA. If using both Protein A and Protein G beads, mix an equal volume of  Protein A and Protein G beads and wash three times in RIPA Buffer.  Aspirate RIPA Buffer and add single stranded herring sperm DNA to a  final concentration of 75 ng/μl beads and BSA to a final concentration  of 0.1 μg/μl beads. Add RIPA Buffer to twice the bead volume and  incubate for 30 min with rotation at RT. Wash once with RIPA Buffer and  add RIPA Buffer to twice the bead volume.

Protein  A beads, protein G beads or a mix of both should be used. Table 1 shows  the affinity of protein A and G beads to different Immunoglobulin  isotypes.

5)      Centrifuge the protein A/G beads for 1 min, 2,000 g and remove the supernatant.

6)      Wash beads three times with 1 ml Wash Buffer. Centrifuge 1 min, 2,000 g and remove the supernatant.

7)      Wash beads one time with 1 ml Final Wash Buffer. Centrifuge 1 min, 2,000 g and remove the supernatant.

If  high background is observed additional washes may be needed.  Alternatively, the sonicated chromatin may also be pre-cleared by  incubating with the Protein A/G beads for 1 hr prior to Step 4.2. Any  non-specific binding to the beads will be removed during this additional  step. Transfer the supernatant (sonicated chromatin) to a new tube and  incubate with the antibody and beads as described in Step 4.2 onwards.

7.        Elution and reverse cross-links

1)      Elute DNA by adding 120 μl of Elution Buffer to the protein A/G beads and rotate for 15 min, 30°C.

2)      Centrifuge for 1 min, 2,000 g and transfer the supernatant into a fresh tube. The samples can be stored at -20°C

3)      The  DNA can be purified using a PCR purification kit (proceed with Step  3.2a) or phenol:chloroform (add 280 μl of Elution Buffer and proceed  with Step 3.2b).

4)      DNA  levels are quantitatively measured by real-time PCR. Primers and probes  are often designed using software provided with the real-time PCR  apparatus.