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Oct 2020
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Histone modification ChIP-seq on Arabidopsis thaliana Plantlets
拟南芥植株上的组蛋白修饰ChIP-seq   

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Abstract

Characterizing the molecular mechanisms regulating gene expression is crucial for understanding the regulatory processes underlying physiological responses to environmental and developmental signals in eukaryotes. The covalent modification of histones contributes to the compaction levels of chromatin, as well as the recruitment of the transcriptional machinery to specific loci, facilitating metastable changes in gene activity. ChIP-seq (Chromatin Immunoprecipitation followed by sequencing) has become the gold standard method for determining histone modification profiles among different organisms, tissues, and genotypes. In the current protocol, we describe a highly robust method for performing ChIP-seq of histone modifications in Arabidopsis thaliana plantlets. Besides its robustness, this method uses in-house-prepared buffers for chromatin extraction, immunoprecipitation, washing, and elusion, making it cost-effective in contrast to commercial kits.

Keywords: Chromatin (染色质), Immunoprecipitation (免疫沉淀), ChIP-seq (结合位点分析法), Histone ( 组蛋白), Arabidopsis thaliana ( 拟南芥)

Background

In eukaryotes, particular histone modifications can be associated with specific transcriptional status (Dong and Weng, 2013). Thus, integrating data on chromatin modification patterns with the transcriptional landscape of specific genotypes and tissues, as well as in response to treatments, can contribute with valuable information for the prediction of transcriptional networks regulating a plethora of physiological processes. ChIP-seq exploits the high specificity of immunoassays with the high-throughput nature of next-generation sequencing (NGS), representing a robust, replicable, and cost-effective technique. This method has been equally adapted for the identification of genomic targets of chromatin-interacting proteins, including transcription factors, allowing expanding its applications to practically any research field of biological sciences.

Materials and Reagents

  1. 50 ml Falcon tubes

  2. Plastic film strips (6 × 2.5 cm)

  3. 100 μm filters

  4. 1.5 ml tube-adapted rotator mixer

  5. ChIP-compatible antibodies (references)

    Anti-H3K27me3 (Millipore, catalog number: 07-449)

    Anti-H3K27me1 (Millipore, catalog number: 07-448)

    Anti-H3K9ac (Millipore, catalog number: 07-352)

    Anti-H3K14ac (Millipore, catalog number: 07-353)

    Anti-H3K4me3 (Millipore, catalog number: 07-473)

  6. Formaldehyde solution 37% (Sigma-Aldrich, catalog number: 252549)

  7. Glycine (Sigma-Aldrich, catalog number: 410225)

  8. Sucrose ≥99.5% (GC) (Sigma-Aldrich, catalog number: S9378)

  9. Tris-HCl (Sigma-Aldrich, catalog number: T3253)

  10. MgCl2 (Sigma-Aldrich, catalog number: M2670)

  11. 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250)

  12. cOmpleteTM, EDTA-free Protease Inhibitor Cocktail (Roche, Sigma-Aldrich, catalog number: COEDTAF-RO)

  13. TritonTM X-100 (Sigma-Aldrich, catalog number: X100)

  14. Ethylenediaminetetraacetic acid (EDTA) 0.5 M solution (Sigma-Aldrich, catalog number: 03690)

  15. NaCl (Sigma-Aldrich, catalog number: S7653)

  16. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: 436143)

  17. LiCl (Sigma-Aldrich, catalog number: 310468)

  18. IGEPAL® CA-630 (Supelco, Sigma-Aldrich, catalog number: 56741)

  19. NaHCO3 (Sigma-Aldrich, catalog number: S8875)

  20. Phenol:Chloroform:Isoamyl alcohol (Sigma-Aldrich, catalog number: P2069)

  21. Sodium Acetate Solution 3 M (Sigma-Aldrich, catalog number: 71196)

  22. Dynabeads Protein A or G (ThermoFisher, Invitrogen, catalog number: 10001D/10003D)

  23. GlycoBlueTM Coprecipitant (ThermoFisher, Invitrogen, catalog number: AM9516)

  24. Proteinase K Solution (ThermoFisher, Invitrogen, catalog number: 25530049)

  25. RNase CocktailTM Enzyme Mix (ThermoFisher, Invitrogen, catalog number: AM2286)

  26. QubitTM dsDNA HS Assay Kit (ThermoFisher, Invitrogen, catalog number: Q32851)

  27. Glycerol

  28. Liquid nitrogen

  29. 100% ethanol, absolute

  30. Sterile Milli-Q water

  31. Extraction Buffer 1 (see Recipes)

  32. Extraction Buffer 2 (see Recipes)

  33. Extraction Buffer 3 (see Recipes)

  34. Nuclei Lysis Buffer (see Recipes)

  35. ChIP Dilution Buffer (see Recipes)

  36. Low Salt Wash Buffer (see Recipes)

  37. High Salt Wash Buffer (see Recipes)

  38. LiCl Wash Buffer (see Recipes)

  39. TE Buffer (see Recipes)

  40. Elution Buffer (see Recipes)


Solutions for buffer preparation (see Recipes)
  1. Glycine 2 M (autoclave)

  2. Tris-HCl pH 8 1 M (autoclave)

  3. Tris-HCl pH 6.5 1 M (autoclave)

  4. Sucrose 2 M (autoclave)

  5. MgCl2 1 M (autoclave)

  6. NaCl 5 M (autoclave)

  7. LiCl 4 M (autoclave)

  8. Igepal CA-630 10% (do not autoclave)

  9. Triton X-100 20% (do not autoclave)

  10. Glycerol 30% (do not autoclave)

Equipment

  1. Vacuum Desiccator

  2. Mortars and pestles

  3. Funnel

  4. Refrigerated Microcentrifuge

  5. Small paintbrush

  6. Focused-Ultrasonicator (Covaris, model: S220)

  7. Safe-Lock microcentrifuge tubes (Eppendorf®)

  8. Water bath or heat block

  9. Agarose for electrophoresis gel

  10. Magnetic rack for 1.5 ml tubes

  11. Mini spin centrifuge

  12. Qubit Fluorometer (ThermoFisher, Invitrogen)

Software

  1. The pipeline used for ChIP-seq analysis is presented in the original paper where this method has been used (Antunez-Sanchez et al., 2020: https://elifesciences.org/articles/58533).

Procedure

A schematic diagram depicting the whole experimental procedure is shown in Figure 1.




Figure 1. Experimental overview. A. Plant tissue is crosslinked to preserve protein-DNA interactions. B. After crosslinking, chromatin is extracted from the tissue through grinding and using a series of extraction buffers. C-D. Extracted chromatin is sonicated to obtain DNA fragments of the desired size (150-500 bp). Sonication is validated by visualizing the DNA in an agarose gel. E. Sonicated chromatin is immunoprecipitated with the chosen antibodies and non-enriched chromatin washed with a series of washing buffers. The input is prepared. Finally, immunoprecipitated DNA is de-crosslinked from interacted proteins and eluted. Eluted DNA can be used for NGS library preparation.


  1. Crosslinking of plant tissue (perform under extraction hood)

    1. Collect 14-day-old plantlets grown in ½ MS. Add approximately 3 g of tissue to a 50 ml Falcon tube containing 36 ml of water (divide samples in different tubes if necessary).

    2. Add 1 ml of 37% Formaldehyde stock to each Falcon tube (for a final concentration of 1%).

    3. Close tubes and invert several times to mix.

    4. Remove tube lids. Add one filmstrip to each tube, pushing it down until it touches the top of the solution (Figure 2). This prevents the plants from floating out of the liquid during the vacuum infiltration.



      Figure 2. Schematic representation of the vacuum infiltration setup for crosslinking. A plastic filmstrip is added to each tube to ensure that the plant tissue stays submerged during vacuum infiltration.


    5. Rapidly proceed to vacuum infiltrate the samples for 15 min.

    6. After 15 min, add 2.5 ml of the glycine 2 M solution, close the tubes, and mix by inversion.

    7. Open the tubes and vacuum infiltrate for an additional 5 min (to quench the formaldehyde).

    8. Strain the samples to remove the crosslinking solution, disposing of it in the appropriate chemical waste container.

    9. Rinse with running distilled water until the crosslinking solution is completely washed away.

    10. Remove the excess water from each sample by placing it on absorbing paper and pressing it gently.

    11. Place the crosslinked tissue on an aluminum foil square and fold it to create a pouch. Label each sample and snap freeze them in liquid nitrogen.

    12. If desired, the experiment can be stopped at this step and samples stored at -80°C. Otherwise, continue to chromatin extraction.


  2. Chromatin extraction (perform on ice)

    1. Cool-down Falcon- and Eppendorf-adapted centrifuges to 4°C.

    2. Prepare Extraction Buffer 1, Extraction Buffer 2, and Extraction Buffer 3 (these buffers can be stored at 4°C prior to the addition of β-mercaptoethanol and protease inhibitors). Prepare Nuclei Lysis buffer (prepare fresh each time).

    3. Add β-mercaptoethanol and protease inhibitors to Extraction Buffer 1, Extraction Buffer 2, and Extraction Buffer 3 to obtain the desired final concentrations (see materials and reagents).

    4. Prepare and label a 50 ml Falcon tube per sample, remove the cap, and place it on ice.

    5. Add liquid nitrogen to a mortar to cool it down. Grind samples thoroughly with mortar and pestle, adding liquid nitrogen constantly to avoid thawing. As soon as the sample is completely ground, place the powdered tissue in the labeled Falcon tube, close the tube, and place it in liquid nitrogen.

    6. As soon as all samples are ground, place falcons with powdered tissue on ice and add 25 ml of Extraction Buffer 1 to each tube. Mix samples slowly by inversion until the solution is homogeneous; open the tubes several times during this step to avoid gas accumulation.

    7. Prepare and label a new set of 50 ml Falcon tubes on ice. Filter each sample into a new Falcon using 100 μm-metal filters placed in a funnel (rinse the funnel and filter between each sample with distilled water).

    8. Centrifuge filtrates at 1,500 × g for 15 min in a pre-cooled centrifuge.

    9. Gently remove the supernatant with a 25 ml pipette or by slowly inverting the tubes on the appropriate chemical waste container. Be careful not to lose pellet material.

    10. Carefully add 20 ml of cold Extraction Buffer 2 to each tube and resuspend the pellet gently using a paintbrush. After most of the pellet is resuspended, close the Falcon tubes, invert slowly, and incubate on ice for 5-10 min. Wash the paintbrush between samples.

    11. Centrifuge at 1,500 × g for 10 min in a pre-cooled centrifuge (4°C).

    12. Gently remove supernatant using a 25 ml pipette without disturbing the pellet.

    13. Resuspend the pellet in 500 μl of Extraction Buffer 3 (stir with a paintbrush) and gently pipet up and down a few times, avoiding creating air bubbles (samples are delicate). If samples are too thick after resuspending them, add another 500 μl of Extraction Buffer 3.

    14. Prepare two 1.5 ml Eppendorf tubes per sample and place them on ice. Add 500 μl of Extraction Buffer 3 on each tube (if an extra volume of Extraction Buffer 3 has been added on Step B13, prepare one extra Eppendorf tube/sample).

    15. Delicately place each sample from step 13 on top of the two (or three) tubes prepared on step 14 (containing 500 μl of Extraction Buffer 3). The buffer will be used to create a sugar gradient that will allow filtering the samples.

    16. Centrifuge at 16,000 × g for 1 h in a pre-cooled centrifuge (4°C).

    17. Prepare fresh Nuclei Lysis (can be kept ON or for a few days in the fridge) and ChIP dilution Buffer (can be kept a few weeks in the fridge).

    18. Discard the supernatant from Step B16.

    19. Resuspend each pellet in 300 μl of Nuclei Lysis Buffer (if the sample is too viscous, add 100 μl extra).

    20. Regroup all the samples for the same genotype/treatment in a single cold 1.5 ml Eppendorf tube. If the final volume of chromatin per sample is less than 1 ml, complete to this volume with Nuclei Lysis Buffer.

    21. If desired, the experiment can be stopped at this step and samples stored at -20°C. Otherwise, continue directly to chromatin sonication.


  3. Chromatin sonication (perform all steps on ice)

    1. Turn on Covaris Sonicator (it takes 20-30 min to cool down and degas).

    2. Transfer 1 ml of chromatin in Covaris 1 ml tubes with AFA fiber.

    3. Sonicate samples (program: Peak Incident Power = 175 W, Duty Factor = 20, cycle/burst = 200, and duration = 5 min).

    4. Keep samples in Covaris tubes on ice and proceed to validate the chromatin sonication. Sonicated chromatin with debris can be stored at -20°C.


  4. Validation of sonication quality (perform all steps on ice)

    1. Place 60 μl of each sonicated chromatin into 1.5 ml Safe-Lock Eppendorf tubes and centrifuge for 5 min (16,000 × g, 4°C).

    2. Transfer 50 μl of each supernatant into a new Eppendorf tube and add 1 μl of RNase A + T1. Incubate at 37°C for 20 min.

    3. Add 2 μl of 5M NaCl + 1 μl of 0.5 M EDTA + 2 μl of 1 M Tris-HCl pH 6.5 + 1 μl Proteinase K to each sample. Incubate at 65°C for 1 h.

    4. Spin down each sample briefly.

    5. Add 114 μl of Phenol/Chloroform/IAA to each sample (under the laminar hood), close tightly, and vortex for 30 s.

    6. Centrifuge at 13,000 × g for 5 min (room temperature).

    7. Transfer the supernatant (~ 50 μl) to a new 1.5 ml tube and add an extra 1 μl of RNase A + T1 to each sample to completely eliminate RNAs. Incubate at 37°C for 20 min.

    8. Mix 25 μl of each sample from Step D7 with 6 μl of 30% glycerol and load it on a 1% agarose gel, using a 100-1,000 bp ladder. (Glycerol replaces loading buffer).

    9. Run the gel at 100 V for 20 min.

    10. Visualize gel and check that DNA size ranges from 150-500 bp (Figure 3A-3C). If sonication worked as expected, proceed to prepare inputs and to the immunoprecipitation. If sonicated DNA size is larger than needed (Figure 3D, 3E), re-sonicate chromatin samples from Step C4 and repeat the validation of sonication.



      Figure 3. Agarose gel of sonicated Arabidopsis chromatin. Samples in lanes A, B, and C are sufficiently sonicated. Samples in lanes D and E need further sonication before immunoprecipitation.


  5. Immunoprecipitation and Input preparation

    1. Prepare ChIP Dilution Buffer, Low Salt Wash Buffer, High Salt Buffer, LiCl wash Salt Buffer, and TE buffer (can be stored at 4°C prior to the addition of protease inhibitors).

    2. Transfer chromatin from the Covaris tube to 1.5 ml tubes and centrifuge at 16,000 × g 5 min at 4°C.

    3. Transfer supernatant with chromatin into new tubes.

    4. Transfer 10 μl of each sample of sonicated chromatin to be used as the input (store at -20°C).

    5. For each immunoprecipitation, add 200 μl of sonicated chromatin to a 1.5 ml Safe-Lock tube and add 600 μl of cold ChIP dilution buffer. On average, three immunoprecipitations are set for every chromatin sample.

    6. Add desired antibodies to each immunoprecipitation tube (the general standard for antibodies against histone modifications is 3 μg/reaction). Close lid well, invert tube several times to mix, and leave mixing ON in a rotator mixer at 4°C.

    7. Next day, prepare 40 μl of Dynabeads (A for rabbit and G for mouse antibodies) per each immunoprecipitation sample in a 1.5 ml Eppendorf tube. Mix, spin quickly, and place the tube on the magnetic rack to separate beads from the supernatant. Remove supernatant while the tube is on the rack and repeat for a total of three washes with ChIP Dilution Buffer.

    8. Resuspend the beads in 40 μl of ChIP dilution buffer per sample.

    9. Add 40 μl of washed beads to each immunoprecipitated sample. Invert tubes to mix and place samples back in rotation at 4°C for at least 2 h in a lab rotator mixer.

    10. Quickly spin the samples and place tubes on the magnetic rack until beads and supernatant are separated.

    11. Remove supernatant by pipetting while the tubes stand on the magnetic rack.

    12. Add 1 ml of Low Salt Wash Buffer to each sample, close tubes, remove from the magnetic rack, and invert to mix. Quickly spin the tubes and place them back on the magnetic rack until beads and supernatant are separated. Remove supernatant using a pipette while keeping the tubes on the rack.

    13. Add 1 ml of Low Salt Wash Buffer to each sample, close tubes, and remove from the magnetic rack. Incubate 5 min at 4°C on a rotating wheel. Quickly spin the tubes and place them back on the magnetic rack until beads and supernatant are separated. Remove supernatant with a pipette while keeping the tubes on the rack.

    14. Repeat Steps E12 and E13 with 1 ml of High Salt Buffer.

    15. Repeat Steps E12 and E13 with 1 ml of LiCl wash Salt Buffer.

    16. Repeat Steps E12 and E13 with 1 ml of TE buffer. Remove supernatant with pipette, and remove tubes from the magnetic rack.

    17. Prepare Elution Buffer.

    18. Add 200 μl of Elution Buffer, vortex, and incubate at 65°C for 15 min (vortex every 2-3 min).

    19. Quickly spin and place the tubes on a magnetic rack until the beads and supernatant are completely separated.

    20. Transfer supernatants into new 1.5 ml Eppendorf tubes while keeping the tubes with the beads on the magnetic rack.

    21. Repeat Steps E18 and E19, and combine the supernatants into a single 1.5 ml Eppendorf tube for a final volume of 400 μl.

    22. Take input samples from the freezer (10 μl) and add 390 μl of Elution Buffer. From this step, process inputs the same way as immunoprecipitations.

    23. Add 16 μl of 5M NaCl and incubate at 65°C overnight to reverse crosslink.

    24. Quickly spin and add 1 μl of RNase A+T1. Incubate at 37°C for 20 min.

    25. Add 8 μl of 0.5 M EDTA + 16 μl of 1 M Tris-HCl pH 6.5 + 4 μl Proteinase K to each sample. Incubate at 65°C for 3 h.

    26. Spin down briefly in a bench spin centrifuge to collect samples on the bottom of the tubes.

    27. Add 450 μl of Phenol/Chloroform/IAA to each sample (under the laminar hood), close tightly, and vortex for 30 s (use Safelock tubes).

    28. Centrifuge at 13,000 × g for 10 min (room temperature).

    29. Transfer the supernatant (approximately 400 μl) of each sample into new tubes. Add 40 μl of 3M sodium acetate pH 5.5 + 1 μl Glycoblue. Mix by inverting and quickly spin. Add 1 ml of cold 100% ethanol, mix by inversion, and place at -20°C ON to precipitate DNA.

    30. Centrifuge at 14,000 × g for 20 min (4°C).

    31. Remove and discard the supernatant without disturbing the blue pellet. Wash pellet with 700 μl of 70% ethanol.

    32. Centrifuge at maximum speed for 10 min (4°C).

    33. Carefully remove the ethanol without disturbing the pellet. Allow the pellet to dry either on the bench (10 min) or under vacuum (5 min).

    34. Resuspend the pellet in 10-20 μl of water (depending on the number of immunoprecipitations performed for each sample and taking into account that the maximum volume that can be used to produce DNA libraries with a NEBNext Kit is 50 μl).

    35. Pull together the immunoprecipitations corresponding to the same sample.

    36. Quantify the ChIPed DNA for each sample and input with the Qubit dsDNA High Sensitivity Assay Kit. For ChIP-seq, each library should be prepared from 5-10 ng of DNA (ideally 10 ng), using the NEBNext Ultra II DNA Library Prep Kit or equivalent (10-12 cycles on the PCR step). If ChIPed DNA is lower than 10 ng per sample, it is recommended to repeat the procedure from Step E5 with an additional 200 μl of chromatin and pool the resulting IPs together.

Data analysis

The pipeline used for ChIP-seq analysis is presented in the original paper where this method has been used (Antunez-Sanchez et al., 2020: https://elifesciences.org/articles/58533).

Recipes

Stock Solutions:

  1. Glycine 2 M (autoclave)

    Composition      For 100 ml
    Glycine     15 g
    H2O     Complete to 100 ml

  2. Tris-HCl pH 8, 1 M (autoclave)

    Composition      For 100 ml
    Tris-HCl     12.114 g
    H2O     Complete to 80 ml, adjust pH to 8 with NaOH, and then complete to 100 ml

  3. Tris-HCl pH 6.5, 1 M (autoclave)

    Composition      For 100 ml
    Tris-HCl     12.114 g
    H2O     Complete to 80 ml, adjust pH to 6.5 with NaOH, and then complete to 100 ml

  4. Sucrose, 2 M (autoclave)

    Composition      For 500 ml
    Sucrose     342.2965 g
    H2O     Complete to 500 ml

  5. MgCl2, 1 M (autoclave)

    Composition For 100 ml
    MgCl2                       9.5211 g (or 20.33 g if hexahydrate)
    H2O Complete to 100 ml

  6. NaCl, 5 M (autoclave)

    Composition      For 100 ml
    NaCl     29.22 g
    H2O     Complete to 100 ml

  7. LiCl, 4 M (autoclave)

    Composition      For 50 ml
    LiCl     8.5 g
    H2O     Complete to 50 ml

  8. Igepal CA-630 10% (do not autoclave)

    Composition      For 50 ml
    Igepal CA-630     5 ml
    H2O     45 ml

  9. Triton X-100 20% (do not autoclave)

    Composition      For 50 ml
    Triton X-100     10 ml
    H2O     40 ml

  10. Glycerol 30% (do not autoclave)

    Composition      For 10 ml
    Glycerol     3.33 ml
    H2O     6.66 ml

  11. Extraction Buffer 1

    Note: It can be stored at 4°C before adding protease inhibitors and 2- mercaptoethanol.

    Composition     For 100 ml
    0.4 M Sucrose     20 ml of 2 M solution
    10 mM Tris-HCl pH 8     1 ml of 1 M solution
    10 mM MgCl2     1 ml of 1 M solution
    5 mM 2-mercaptoethanol      35 μl of 14.4 M solution
    Protease Inhibitors     200 μl of 1 tablet/ml solution
    H2O     77.8 ml

  12. Extraction Buffer 2

    Note: It can be stored at 4°C before adding protease inhibitors and 2-mercaptoethanol.

    Composition     For 100 ml
    0.25 M Sucrose     12.5 ml of 2 M solution
    10 mM Tris-HCl pH 8     1 ml of 1 M solution
    10 mM MgCl2     1 ml of 1 M solution
    1% Triton X-100     5 ml of 20% solution
    5 mM 2-mercaptoethanol      35 μl of 14.4 M solution
    Protease Inhibitors     200 μl of 1 tablet/ml solution
    H2O     80.3 ml

  13. Extraction Buffer 3

    Note: It can be stored at 4°C before adding protease inhibitors and 2-mercaptoethanol.

    Composition     For 100 ml
    1.7 M Sucrose     85 ml of 2 M solution
    10 mM Tris-HCl pH 8     1 ml of 1 M solution
    2 mM MgCl2     200 μl of 1 M solution
    0.15% Triton X-100     750 μl of 20% solution
    5 mM 2-mercaptoethanol      35 μl of 14.4 M solution
    Protease Inhibitors     200 μl of 1 tablet/ml solution
    H2O     12.8 ml

  14. Nuclei Lysis Buffer (prepare fresh each time)

    Composition             For 5 ml
    50 mM Tris-HCl pH 8              250 μl of 1M solution
    10 mM EDTA             100 μl of 0.5M solution
    0.1% SDS             25 μl of 20% solution
    Protease inhibitors             100 μl of 1 tablet/ml solution
    H2O             4.525 ml

  15. ChIP Dilution Buffer

    Note: It can be stored at 4°C before adding protease inhibitors.

    Composition         For 100 ml
    1.1% Triton X-100         5.5 ml of 20% solution
    1.2 mM EDTA         240 μl of 0.5 M solution
    16.7 mM Tris-HCl pH 8          1.67 ml of 1 M solution
    167 mM NaCl         3.34 ml of 5 M solution
    Protease inhibitors         200 μl of 1 tablet/ml solution
    H2O         89.05 ml

  16. Low Salt Wash Buffer

    Note: It can be stored at 4°C.

    Composition         For 100 ml
    150 mM NaCl         1.5 ml of 5M solution
    0.1% SDS         250 μl of 20% solution
    1% Triton X-100         2.5 ml of 20% solution
    2 mM EDTA         200 μl of 0.5 M solution
    20 mM Tris-HCl pH 8          1 ml of 1 M solution
    H2O         44.3 ml

  17. High Salt Wash Buffer

    Note: It can be stored at 4°C.

    Composition         For 100 ml
    500 mM NaCl         5 ml of 5 M solution
    0.1% SDS         250 μl of 20% solution
    1% Triton X-100         2.5 ml of 20% solution
    2 mM EDTA         200 μl of 0.5 M solution
    20 mM Tris-HCl pH 8          1 ml of 1 M solution
    H2O         41.05 ml

  18. LiCl Wash Buffer

    Note: It can be stored at 4°C.

    Composition         For 50 ml
    0.25 M LiCl         3.125 ml of 4 M solution
    1% Igepal CA630         5 ml of 10% solution
    1 mM EDTA         100 μl of 0.5 M solution
    10 mM Tris-HCl pH 8          500 μl of 1 M solution
    H2O         41.25 ml

  19. TE Buffer

    Note: It can be stored at 4°C.

    Composition         For 50 ml
    1 mM EDTA         100 μl of 0.5 M solution
    10 mM Tris-HCl pH 8          500 μl of 0.5 M solution
    H2O         49.4 ml

  20. Elution Buffer (prepare fresh each time)

    Composition                     For 20 ml
    1% SDS                     1 ml of 20% solution
    0.1 M NaHCO3                     168 g
    H2O                     19 ml

Acknowledgments

This work was funded by the Agence National de la Recherche ANR (3DWheat project ANR-19-CE20-0001-01) and by the Institut Universitaire de France (IUF).

This protocol was derived from previously published research articles (Ramirez-Prado et al., 2019; Antunez-Sanchez et al., 2020; Concia et al., 2020).

Competing interests

The authors declare that no competing interests exist.

References

  1. Antunez-Sanchez, J., Naish, M., Ramirez-Prado, J. S., Ohno, S., Huang, Y., Dawson, A., Opassathian, K., Manza-Mianza, D., Ariel, F. and Raynaud, C. (2020). A new role for histone demethylases in the maintenance of plant genome integrity. Elife 9: e58533.
  2. Concia, L., Veluchamy, A., Ramirez-Prado, J. S., Martin-Ramirez, A., Huang, Y., Perez, M., Domenichini, S., Rodriguez Granados, N. Y., Kim, S. and Blein, T. (2020). Wheat chromatin architecture is organized in genome territories and transcription factories. Genome Biol 21(1): 104.
  3. Dong, X. and Weng, Z. (2013). The correlation between histone modifications and gene expression. Epigenomics 5(2): 113-116.
  4. Ramirez-Prado, J. S., Latrasse, D., Rodriguez-Granados, N. Y., Huang, Y., Manza-Mianza, D., Brik-Chaouche, R., Jaouannet, M., Citerne, S., Bendahmane, A. and Hirt, H. (2019). The Polycomb protein LHP1 regulates Arabidopsis thaliana stress responses through the repression of the MYC2-dependent branch of immunity. Plant J 100(6): 1118-1131.

简介

[抽象的]表征调控基因表达的分子机制对于理解真核生物对环境和发育信号的生理反应的潜在调控过程至关重要。组蛋白的共价修饰有助于染色质的压实水平,以及将转录机制募集到特定位点,促进基因活性的亚稳态变化。ChIP-seq(染色质免疫沉淀随后测序)已成为确定不同生物体、组织和基因型之间组蛋白修饰谱的金标准方法。在当前的协议中,我们描述了一种在拟南芥中进行组蛋白修饰的 ChIP-seq 的高度稳健的方法小苗。除了其稳健性之外,该方法还使用内部制备的缓冲液进行染色质提取、免疫沉淀、洗涤和洗脱,与商业试剂盒相比,具有成本效益。

[背景] 在真核生物中,特定的组蛋白修饰可能与特定的转录状态相关(Dong 和 Weng,2013)。因此,将染色质修饰模式的数据与特定基因型和组织的转录景观以及对治疗的反应相结合,可以为预测调节过多生理过程的转录网络提供有价值的信息。ChIP-seq 利用免疫测定的高特异性和下一代测序 (NGS) 的高通量特性,代表了一种稳健、可复制且具有成本效益的技术。该方法同样适用于鉴定染色质相互作用蛋白的基因组靶标,包括转录因子,从而使其应用扩展到几乎任何生物科学研究领域。

关键字:染色质, 免疫沉淀, 结合位点分析法, 组蛋白, 拟南芥


材料和试剂

 
50 毫升猎鹰管
塑料薄膜条(6 × 2.5 cm)
100 μ米过滤器
1.5 ml 管式旋转混合器
ChIP 兼容抗体(参考文献)
Anti-H3K27me3(Millipore,目录号:07-449)
Anti-H3K27me1(Millipore,目录号:07-448)
Anti-H3K9ac(Millipore,目录号:07-352)
Anti-H3K14ac(Millipore,目录号:07-353)
Anti-H3K4me3(Millipore,目录号:07-473)
甲醛溶液37%(Sigma-Aldrich,目录号:252549)
甘氨酸(Sigma-Aldrich,目录号:410225)
蔗糖 ≥99.5%(GC)(Sigma-Aldrich,目录号:S9378)
Tris-HCl(Sigma-Aldrich,目录号:T3253)
MgCl 2 (Sigma-Aldrich,目录号:M2670)
2-巯基乙醇(Sigma-Aldrich,目录号:M6250)
cOmplete TM ,无 EDTA 蛋白酶抑制剂混合物(Roche,Sigma-Aldrich,目录号:COEDTAF-RO)
Triton TM X-100(Sigma-Aldrich,目录号:X100)
乙二胺四乙酸(EDTA)0.5 M溶液(Sigma-Aldrich,目录号:03690)
NaCl(Sigma-Aldrich,目录号:S7653)
十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:436143)
LiCl(Sigma-Aldrich,目录号:310468)
IGEPAL ® CA-630(Supelco公司,Sigma-Aldrich公司,目录号:56741)
NaHCO 3 (Sigma-Aldrich,目录号:S8875)
苯酚:氯仿:异戊醇(Sigma-Aldrich,目录号:P2069)
醋酸钠溶液3 M(Sigma-Aldrich,目录号:71196)
Dynabeads Protein A 或 G(ThermoFisher,Invitrogen,目录号:10001D/10003D)
GlycoBlue TM共沉淀剂(ThermoFisher,Invitrogen,目录号:AM9516)
蛋白酶 K 溶液(ThermoFisher,Invitrogen,目录号:25530049)
RNase Cocktail TM Enzyme Mix(ThermoFisher,Invitrogen,目录号:AM2286)
Qubit TM dsDNA HS 检测试剂盒(ThermoFisher,Invitrogen,目录号:Q32851)
甘油
液氮
100% 乙醇,绝对
无菌 Milli-Q 水
提取缓冲液 1(见 食谱)
提取缓冲液 2(见配方)
提取缓冲液 3(见配方)
细胞核裂解缓冲液(见配方)
ChIP 稀释缓冲液(参见配方)
低盐洗涤缓冲液(见配方)
高盐洗涤缓冲液(见配方)
LiCl 洗涤缓冲液(见配方)
TE 缓冲液(见配方)
洗脱缓冲液(见配方)
 
缓冲液制备溶液(见配方)
甘氨酸 2 M(高压釜)
Tris-HCl pH 8 1 M(高压釜)
Tris-HCl pH 6.5 1 M(高压釜)
蔗糖 2 M(高压釜)
MgCl 2 1 M(高压釜)
NaCl 5 M(高压釜)
LiCl 4 M(高压釜)
Igepal CA-630 10%(不可高压灭菌)
Triton X-100 20%(不要高压灭菌)
甘油 30%(不要高压灭菌)
 
设备
 
真空干燥器                           
研钵和研杵
漏斗
冷冻微量离心机
小画笔
聚焦超声仪(Covaris,型号:S220)
Safe-Lock 微量离心管 (Eppendorf ® )
水浴或加热块
电泳凝胶用琼脂糖
用于 1.5 毫升试管的磁性架
微型离心机
Qubit 荧光计(ThermoFisher,Invitrogen)
 
软件
 
用于 ChIP-seq 分析的管道在使用此方法的原始论文中介绍(Antunez-Sanchez等人,2020 年:https : //elifesciences.org/articles/58533)。
 
程序
 
描绘整个实验过程的示意图如图1所示。
 
 
图 1. 实验概述。A.植物组织被交联以保持蛋白质-DNA 相互作用。B.交联后,通过研磨和使用一系列提取缓冲液从组织中提取染色质。光盘。对提取的染色质进行超声处理以获得所需大小 (150-500 bp) 的 DNA 片段。通过在琼脂糖凝胶中可视化 DNA 来验证超声处理。E.超声处理的染色质用选定的抗体免疫沉淀,未富集的染色质用一系列洗涤缓冲液洗涤。输入已准备好。最后,免疫沉淀的 DNA 从相互作用的蛋白质中去交联并被洗脱。洗脱的 DNA 可用于 NGS 文库制备。
 
植物组织的交联(在提取罩下进行)
收集在 ½ MS 中生长的 14 天龄小苗。将大约 3 克组织加入含有 36毫升水的 50毫升Falcon 管中(如有必要,将样品分在不同的管中)。
向每个 Falcon 管中加入 1 ml 37% 的甲醛储备液(最终浓度为 1%)。
关闭试管并颠倒几次以混合。
取下管盖。在每个管中添加一个胶片,将其向下推,直到它接触到溶液的顶部(图 2)。这可以防止植物在真空渗透过程中从液体中浮出。
 
 
图 2. 用于交联的真空渗透设置的示意图。每个管子上都添加了塑料薄膜带,以确保植物组织在真空浸润过程中保持浸没。
 
快速进行真空渗透样品 15 分钟。
15 分钟后,加入 2.5 ml 甘氨酸 2 M 溶液,关闭管子,倒置混合。
打开管子,再真空渗透 5 分钟(以淬灭甲醛)。
过滤样品以去除交联溶液,将其丢弃在适当的化学废物容器中。
用流动的蒸馏水冲洗,直到交联溶液被完全洗掉。
通过将每个样品放在吸水纸上并轻轻按压,去除每个样品中多余的水。
将交联的纸巾放在铝箔正方形上,折叠起来制成一个小袋。标记每个样品并在液氮中快速冷冻。
如果需要,实验可以在这一步停止,样品储存在 -80°C。否则,继续进行染色质提取。
 
染色质提取(在冰上进行)
将适应 Falcon 和 Eppendorf 的离心机冷却至 4°C。
准备提取缓冲液 1、提取缓冲液 2 和提取缓冲液 3(这些缓冲液可以在加入 β-巯基乙醇和蛋白酶抑制剂之前在 4°C 下储存)。准备细胞核裂解缓冲液(每次准备新鲜)。
将 β-巯基乙醇和蛋白酶抑制剂添加到提取缓冲液 1、提取缓冲液 2 和提取缓冲液 3 中以获得所需的最终浓度(参见材料和试剂)。
为每个样品准备一个 50 毫升的 Falcon 试管并贴上标签,取下盖子,将其放在冰上。
向研钵中加入液氮使其冷却。用研钵和研杵彻底研磨样品,不断加入液氮以避免解冻。一旦样品完全研磨,将粉末组织放入标记的 Falcon 管中,关闭管,并将其放入液氮中。
研磨完所有样品后,将带有粉末组织的猎鹰放在冰上,然后向每个管中加入 25 ml 提取缓冲液 1。通过倒置缓慢混合样品,直到溶液均匀;在此步骤中多次打开管子以避免气体积聚。
在冰上准备并标记一组新的 50 毫升 Falcon 管。使用放置在漏斗中的 100 μm 金属过滤器将每个样品过滤到新的 Falcon 中(用蒸馏水冲洗每个样品之间的漏斗和过滤器)。
在预冷的离心机中以 1,500 × g离心15 分钟。
用 25 ml 移液器或通过在适当的化学废物容器上缓慢倒转管轻轻去除上清液。小心不要丢失颗粒材料。
小心地将 20 ml 冷提取缓冲液 2 添加到每个管中,并使用画笔轻轻地重悬沉淀。大部分沉淀重悬后,关闭 Falcon 管,缓慢倒置,并在冰上孵育 5-10 分钟。在样品之间清洗画笔。
在预冷离心机 (4°C) 中以 1,500 × g离心10 分钟。
使用 25 ml 移液器轻轻去除上清液,而不会干扰沉淀。
将沉淀重悬在 500 μl 提取缓冲液 3(用画笔搅拌)中,轻轻上下移液几次,避免产生气泡(样品很脆弱)。如果样品重悬后太稠,再加入 500 μl Extraction Buffer 3。
每个样品准备两个 1.5 ml Eppendorf 管并将它们放在冰上。在每个试管中添加 500 μl 提取缓冲液 3(如果在步骤 B13 中添加了额外体积的提取缓冲液 3,请准备一个额外的 Eppendorf 试管/样品)。
将步骤 13 中的每个样品小心地放置在步骤 14 中准备的两(或三个)管(含有 500 μl 提取缓冲液 3)的顶部。缓冲区将用于创建一个糖梯度,允许过滤样本。
在预冷离心机 (4°C) 中以 16,000 × g离心1 小时。
准备新鲜的细胞核裂解(可保持开启或在冰箱中保存几天)和 ChIP 稀释缓冲液(可在冰箱中保存数周)。
丢弃步骤 B16 中的上清液。
将每个颗粒重悬在 300 μl 核裂解缓冲液中(如果样品太粘稠,请额外添加 100 μl)。
在单个冷的 1.5 ml Eppendorf 管中重新组合相同基因型/处理的所有样品。如果每个样品的最终染色质体积小于 1 ml,请使用 Nuclei Lysis Buffer 完成此体积。
如果需要,实验可以在这一步停止,样品储存在 -20°C。否则,直接继续染色质超声处理。
 
染色质超声处理(在冰上执行所有步骤)
打开 Covaris Sonicator(冷却和脱气需要 20-30 分钟)。
将 1 ml 染色质转移到带有 AFA 纤维的 Covaris 1 ml 管中。
超声处理样品(程序:峰值入射功率 = 175 W,占空系数 = 20,周期/突发 = 200,持续时间 = 5 分钟)。
将样品保存在冰上的 Covaris 管中,并继续验证染色质超声处理。带有碎片的超声染色质可以储存在 -20°C。
 
超声质量验证(在冰上执行所有步骤)
将 60 μl 每种经超声处理的染色质放入 1.5 ml Safe-Lock Eppendorf 管中并离心 5 分钟(16,000 × g ,4°C)。
将 50 μl 的每种上清液转移到新的 Eppendorf 管中,并加入 1 μl RNase A + T1。在 37°C 下孵育 20 分钟。
向每个样品中加入 2 μl 5M NaCl + 1 μl 0.5 M EDTA + 2 μl 1 M Tris-HCl pH 6.5 + 1 μl 蛋白酶 K。在 65°C 下孵育 1 小时。
简要地降低每个样品的转速。
向每个样品中加入 114 μl 苯酚/氯仿/IAA(在层流罩下),紧紧关闭,涡旋 30 秒。
以 13,000 × g离心5 分钟(室温)。
将上清液 (~ 50 μl) 转移到新的 1.5 ml 管中,并在每个样品中额外添加 1 μl RNase A + T1 以完全消除 RNA。在 37°C 下孵育 20 分钟。
将 25 μl 来自步骤 D7 的每个样品与 6 μl 30% 甘油混合,并使用 100-1,000 bp 阶梯将其加载到 1% 琼脂糖凝胶上。(甘油代替加载缓冲液)。
在 100 V 下运行凝胶 20 分钟。
可视化凝胶并检查 DNA 大小范围从 150-500 bp (图 3A-3C)。如果超声处理按预期工作,则继续准备输入和免疫沉淀。如果超声处理的 DNA 大小大于所需(图 3D、3E),则对步骤 C4 中的染色质样本进行重新超声处理并重复超声处理验证。
 
 
图 3.超声处理的拟南芥染色质的琼脂糖凝胶。泳道 A、B 和 C 中的样品经过充分超声处理。泳道 D 和 E 中的样品在免疫沉淀之前需要进一步超声处理。
 
免疫沉淀和输入准备
准备 ChIP 稀释缓冲液、低盐洗涤缓冲液、高盐缓冲液、LiCl 洗涤盐缓冲液和 TE 缓冲液(可以在加入蛋白酶抑制剂之前在 4°C 下储存)。
将染色质从 Covaris 管转移到 1.5 ml 管中,并在 4°C 下以 16,000 × g离心5 分钟。
将带有染色质的上清液转移到新管中。
转移每个超声染色质样品 10 μl 用作输入(储存在 -20°C)。
对于每次免疫沉淀,将 200 μl 经超声处理的染色质加入 1.5 ml Safe-Lock 管中,并加入 600 μl 冷 ChIP 稀释缓冲液。平均而言,每个染色质样品设置三个免疫沉淀。
将所需抗体添加到每个免疫沉淀管中(针对组蛋白修饰的抗体的一般标准为 3 μg/反应)。盖好盖子,倒转管数次混合,并在 4°C 的旋转混合器中保持混合。
第二天,在 1.5 ml Eppendorf 管中为每个免疫沉淀样品准备 40 μl Dynabeads(A 代表兔抗体,G 代表小鼠抗体)。混合,快速旋转,并将管子放在磁架上,将珠子与上清液分开。将管子放在架子上时取出上清液,然后用 ChIP 稀释缓冲液重复洗涤总共 3 次。
在每个样品的 40 μl ChIP 稀释缓冲液中重新悬浮珠子。
向每个免疫沉淀样品中加入 40 μl 洗涤过的珠子。在实验室旋转混合器中,倒置管以混合并在 4°C 下将样品重新旋转至少 2 小时。
快速旋转样品并将管子放在磁架上,直到珠子和上清液分离。
当管子站在磁性架上时,通过移液去除上清液。
向每个样品中加入 1 ml 低盐洗涤缓冲液,关闭试管,从磁性架上取出,倒置混合。快速旋转试管并将它们放回磁性架上,直到珠子和上清液分离。使用移液器去除上清液,同时将管子放在架子上。
向每个样品中加入 1 ml 低盐洗涤缓冲液,关闭试管,然后从磁性架上取出。在 4°C 下在旋转轮上孵育 5 分钟。快速旋转试管并将它们放回磁性架上,直到珠子和上清液分离。用移液管去除上清液,同时将管子放在架子上。
用 1 ml 高盐缓冲液重复步骤 E12 和 E13。
用 1 ml LiCl 洗涤盐缓冲液重复步骤 E12 和 E13。
用 1 ml TE 缓冲液重复步骤 E12 和 E13。用移液器取出上清液,从磁性架上取出试管。
准备洗脱缓冲液。
加入 200 μl Elution Buffer,涡旋,65°C 孵育 15 分钟(每 2-3 分钟涡旋一次)。
快速旋转并将管子放在磁性架上,直到珠子和上清液完全分离。
将上清液转移到新的 1.5 ml Eppendorf 管中,同时将带有磁珠的管保持在磁架上。
重复步骤 E18 和 E19,将上清液合并到一个 1.5 ml Eppendorf 管中,最终体积为 400 μl。
从冰箱 (10 μl) 中取出输入样品并添加 390 μl 洗脱缓冲液。从这一步开始,过程输入的方式与免疫沉淀相同。
加入 16 μl 5M NaCl 并在 65°C 下孵育过夜以逆转交联。
快速旋转并加入 1 μl RNase A+T1。在 37°C 下孵育 20 分钟。
向每个样品中加入 8 μl 0.5 M EDTA + 16 μl 1 M Tris-HCl pH 6.5 + 4 μl 蛋白酶 K。在 65°C 下孵育 3 小时。
在台式离心机中短暂旋转以收集管底部的样品。
向每个样品中加入 450 μl 苯酚/氯仿/IAA(在层流罩下),紧紧关闭,涡旋 30 秒(使用 Safelock 管)。
以 13,000 × g离心10 分钟(室温)。
将每个样品的上清液(约 400 μl)转移到新管中。添加 40 μl 3M 醋酸钠 pH 5.5 + 1 μl Glycoblue。颠倒混合并快速旋转。加入 1 ml 冷的 100% 乙醇,倒置混合,置于 -20°C ON 以沉淀 DNA。
以 14,000 × g离心20 分钟(4°C)。
在不干扰蓝色颗粒的情况下取出并丢弃上清液。用 700 μl 70% 乙醇洗涤沉淀。
以最大速度离心 10 分钟 (4°C)。
小心地去除乙醇而不干扰颗粒。让颗粒在工作台上(10 分钟)或真空下(5 分钟)干燥。
将沉淀重悬在 10-20 μl 水中(取决于对每个样品进行的免疫沉淀次数,并考虑到可用于使用 NEBNext Kit 生成 DNA 文库的最大体积为 50 μl)。
将对应于同一样品的免疫沉淀放在一起。
使用 Qubit dsDNA 高灵敏度检测试剂盒对每个样品和输入的 ChIPed DNA 进行量化。对于 ChIP-seq,每个文库应使用NEBNext Ultra II DNA 文库制备试剂盒或等效物(PCR 步骤 10-12 个循环)从 5-10 ng DNA(理想情况下为 10 ng)制备。如果每个样品的 ChIPed DNA 低于 10 ng,建议使用额外的 200 μl 染色质重复步骤 E5 中的程序,并将产生的 IP 混合在一起。
 
数据分析
 
用于 ChIP-seq 分析的管道在使用此方法的原始论文中介绍(Antunez-Sanchez等人,2020 年:https : //elifesciences.org/articles/58533)。
 
食谱
 
库存解决方案
甘氨酸 2M(高压釜)
作品
100毫升
甘氨酸
15 克
H 2 O
完成至 100 毫升
Tris-HCl pH 8, 1 M(高压釜)
作品
100毫升
三盐酸盐
12.114 克
H 2 O
加满至 80 ml,用 NaOH 将 pH 调至 8,然后加满至 100 ml
Tris-HCl pH 6.5,1 M(高压釜)
作品
100毫升
三盐酸盐
12.114 克
H 2 O
加满至 80 ml,用 NaOH 将 pH 调至 6.5,然后加满至 100 ml
蔗糖,2 M(高压釜)
作品
500毫升
蔗糖
342.2965 克
H 2 O
完成至 500 毫升
MgCl 2 , 1 M (高压釜)
作品
100毫升
氯化镁2
9.5211 克(或 20.33 克,如果是六水合物)
H 2 O
完成至 100 毫升
 
氯化钠,5 M(高压釜)
作品
100毫升
氯化钠
29.22 克
H 2 O
完成至 100 毫升
LiCl,4 M(高压釜)
作品
50毫升
氯化锂
8.5 克
H 2 O
完成至 50 毫升
Igepal CA-630 10%(不可高压灭菌)
作品
50毫升
Igepal CA-630
5毫升
H 2 O
45 毫升
Triton X-100 20%(不要高压灭菌)
作品
50毫升
海卫 X-100
10毫升
H 2 O
40 毫升
甘油 30%(不要高压灭菌)
作品
10毫升
甘油
3.33 毫升
H 2 O
6.66 毫升
提取缓冲液 1
注意:在加入蛋白酶抑制剂和 2-巯基乙醇之前,它可以在 4°C 下储存。
作品
100毫升
0.4 M 蔗糖
20ml中2M溶液
10 mM Tris-HCl pH 8
1 毫升 1 M 溶液
10 毫米氯化镁2
1 毫升 1 M 溶液
5 mM 2-巯基乙醇
35μl 14.4 M 溶液
蛋白酶抑制剂
200μl 1 片/ml 溶液
H 2 O
77.8 毫升
提取缓冲液 2
注意:在加入蛋白酶抑制剂和 2-巯基乙醇之前,它可以在 4°C 下储存。
作品
100毫升
0.25 M 蔗糖
12.5 毫升 2 M 溶液
10 mM Tris-HCl pH 8
1 毫升 1 M 溶液
10 毫米氯化镁2
1 毫升 1 M 溶液
1% 海卫 X-100
5 毫升 20% 溶液
5 mM 2-巯基乙醇
35 μl 14.4 M 溶液
蛋白酶抑制剂
200 μl 1 片/ml 溶液
H 2 O
80.3 毫升
提取缓冲液 3
注意:在加入蛋白酶抑制剂和 2-巯基乙醇之前,它可以在 4°C 下储存。
作品
100毫升
1.7 M 蔗糖
85 毫升 2 M 溶液
10 mM Tris-HCl pH 8
1 毫升 1 M 溶液
2 毫米氯化镁2
200 μl 1 M 溶液
0.15% 海卫 X-100
750 μl 20% 溶液
5 mM 2-巯基乙醇
35 μl 14.4 M 溶液
蛋白酶抑制剂
200 μl 1 片/ml 溶液
H 2 O
12.8 毫升
细胞核裂解缓冲液(每次准备新鲜)
作品
5毫升
50 mM Tris-HCl pH8
250 μl 1M 溶液
10 mM EDTA
100 μl 0.5M 溶液
0.1% SDS
25 μl 20% 溶液
蛋白酶抑制剂
100 μl 1 片/ml 溶液
H 2 O
4.525 毫升
ChIP 稀释缓冲液
注意:在加入蛋白酶抑制剂之前,它可以在 4°C 下储存。
作品
100毫升
1.1% 海卫 X-100
5.5 毫升 20% 溶液
1.2 mM EDTA
240 μl 0.5 M 溶液
16.7 mM Tris-HCl pH 8
1.67 毫升 1 M 溶液
167 毫米氯化钠
3.34 毫升 5 M 溶液
蛋白酶抑制剂
200 μl 1 片/ml 溶液
H 2 O
89.05 毫升
低盐洗涤缓冲液
注意:它可以在 4°C 下储存。
作品
100毫升
150 毫米氯化钠
1.5 毫升 5M 溶液
0.1% SDS
250 μl 20% 溶液
1% 海卫 X-100
2.5 毫升 20% 溶液
2 mM EDTA
200 μl 0.5 M 溶液
20 mM Tris-HCl pH 8
1 毫升 1M 溶液
H 2 O
44.3 毫升
高盐洗涤缓冲液
注意:它可以在 4°C 下储存。
作品
100毫升
500 毫米氯化钠
5 毫升 5 M 溶液
0.1% SDS
250 μl 20% 溶液
1% 海卫 X-100
2.5 毫升 20% 溶液
2 mM EDTA
200 μl 0.5 M 溶液
20 mM Tris-HCl pH 8
1 毫升 1 M 溶液
H 2 O
41.05 毫升
氯化锂洗涤缓冲液
注意:它可以在 4°C 下储存。
作品
50毫升
0.25 M氯化锂
3.125 毫升 4 M 溶液
1% 伊格帕尔 CA630
5 毫升 10% 溶液
1 mM EDTA
100 μl 0.5 M 溶液
10 mM Tris-HCl pH 8
500 μl 1 M 溶液
H 2 O
41.25 毫升
TE缓冲液
注意:它可以在 4°C 下储存。
作品
50毫升
1.mM EDTA
100 μl 0.5M 溶液
10mM Tris-HCl pH8
500 μl 0.5M 溶液
H 2 O
49.4 毫升
洗脱缓冲液(每次准备新鲜)
作品
20毫升
1% 安全数据表
1 毫升 20% 溶液
0.1 M 碳酸氢钠3
168 克
H 2 O
19毫升
 
致谢
 
这项工作由 Agence National de la Recherche ANR(3DWheat 项目 ANR-19-CE20-0001-01)和法兰西大学 (IUF) 资助。
该协议源自先前发表的研究文章(Ramirez-Prado等人,2019 年;Antunez-Sanchez等人,2020 年;Concia等人,2020 年)。
 
利益争夺
 
作者声明不存在竞争利益。
 
参考
 
Antunez-Sanchez, J., Naish, M., Ramirez-Prado, JS, Ohno, S., Huang, Y., Dawson, A., Opassathian, K., Manza-Mianza, D., Ariel, F.,雷诺,C.,等。(2020)。组蛋白去甲基化酶在维持植物基因组完整性中的新作用。Elife 9:e58533。
Concia, L., Veluchamy, A., Ramirez-Prado, JS, Martin-Ramirez, A., Huang, Y., Perez, M., Domenichini, S., Rodriguez Granados, NY, Kim, S., Blein, T.,等。(2020)。小麦染色质结构组织在基因组区域和转录工厂中。基因组生物学21(1):104。              
Dong, X. 和 Weng, Z. (2013)。组蛋白修饰与基因表达之间的相关性。 表观基因组学5(2): 113-116。             
Ramirez-Prado, JS, Latrase, D., Rodriguez-Granados, NY, Huang, Y., Manza-Mianza, D., Brik-Chaouche, R., Jaouannet, M., Citerne, S., Bendahmane, A. , Hirt, H.,等。(2019)。Polycomb 蛋白 LHP1通过抑制 MYC2 依赖的免疫分支来调节拟南芥应激反应。 植物学杂志100(6):1118-1131。             

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Copyright Ramirez-Prado et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Ramirez-Prado, J. S., Latrasse, D. and Benhamed, M. (2021). Histone modification ChIP-seq on Arabidopsis thaliana Plantlets. Bio-protocol 11(21): e4211. DOI: 10.21769/BioProtoc.4211.
  2. Antunez-Sanchez, J., Naish, M., Ramirez-Prado, J. S., Ohno, S., Huang, Y., Dawson, A., Opassathian, K., Manza-Mianza, D., Ariel, F. and Raynaud, C. (2020). A new role for histone demethylases in the maintenance of plant genome integrity. Elife 9: e58533.
提问与回复

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满德 薛
IGDB,CAS
Tris-HCl pH 8, 1 M (autoclave) H2O Complete to 80 ml, adjust pH to 8 with NaOH, and then complete to 100 ml above information is wrong, Tris-HCl pH 8 should adjust pH to 8 with HCl not NaOH
11/21/2021 2:09:14 PM 回复
Juan Ramirez-Prado
Centre of Microbial and Plant Genetics (CMPG),KU Leuven, Center for Plant Systems Biology, Leuven

Hello. This is not wrong because the pH of Tris-HCl by default is lower than 8. Hence, adding NaOH will allow the pH to raise to 8.
When you start with Tris base you have to add HCl to decrease the pH.

11/22/2021 12:11:49 AM 回复