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Chromatin Immunoprecipitation Experiments from Whole Drosophila Embryos or Larval Imaginal Discs
整个果蝇胚胎或幼虫成虫盘的染色质免疫沉淀实验   

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Abstract

Chromatin Immunoprecipitation coupled either to qPCR (qChIP) or high-throughput sequencing (ChIP-Seq) has been extensively used in the last decades to identify the DNA binding sites of transcription factors or the localization of various histone marks along the genome. The ChIP experiment generally includes 7 steps: collection of biological samples (A), cross-linking proteins to DNA (B), chromatin isolation and fragmentation by sonication (C), sonication test (D), immunoprecipitation with antibodies against the protein or the histone mark of interest (E), DNA recovery (E), identification of factor-associated DNA sequences by PCR or sequencing (F). The protocol described here can readily be used for ChIP-seq and ChIP-qPCR experiments. The entire procedure, describing experimental setup conditions to optimize assays in intact Drosophila tissues, can be completed within four days.

Keywords: ChIP(ChIP), Drosophila(果蝇), Embryo(胚胎), Imaginal disc(成虫盘), Epigenetic mark(表观遗传标记), Transcription factor(转录因子)

Background

Despite the fact that immortalized cultured cells are extensively used to study the chromatin landscape of various cell types, valuable methods for probing interaction in vivo, under physiological conditions, are necessary to perform temporal or spatial comparative analysis of transcription factor and histone modification maps between different stages of Drosophila development or between different tissues. Here we present a detailed ChIP protocol that has been optimized to work on whole Drosophila embryos and larval imaginal discs, highlighting critical experimental parameters.

Materials and Reagents

  1. Imaginal discs dissection step
    1. Sterile tips for micropipettes, 1,000 µl
    2. 1.5 ml DNA low binding tubes (Eppendorf, catalog number: 022431021 )
    3. Schneider’s insect medium (Sigma-Aldrich, catalog number: S0146 )

  2. Embryos collection step
    1. Petri dishes
    2. 1.5 ml DNA low binding (LoBind) tubes (Eppendorf, catalog number: 022431021 )
    3. Drosophila embryos
    4. SAF-INSTANT yeast (Lesaffre)
    5. Liquid nitrogen
    6. Agar
    7. 10% Moldex (Methyl-4-hydroxybenzoate) (Sigma-Aldrich, catalog number: W271004 ) dissolved in water
    8. Neutral red (Sigma-Aldrich, catalog number: N4638 )
    9. Egg laying medium (see Recipes)

  3. Chromatin immunoprecipitation protocol
    1. 1.5 ml DNA low binding (LoBind) tubes (Eppendorf, catalog number: 022431021 )
    2. 15 ml sterile plastic tubes (Greiner Bio One International, catalog number: 188261 )
    3. 50 ml sterile plastic tubes (Greiner Bio One International, catalog number: 227261 )
    4. 15 ml polystyrene Falcon tubes (Corning, catalog number: 352095 )
    5. Sterile filter tips, 10 µl, 200 µl, 1,000 µl (STARLAB INTERNATIONAL, catalog numbers: S1121-2710 , S1120-8810 , S1122-1830 ) (see Note 1 for more details)
    6. Sterile, DNase and RNase free, DNA low binding filter tips, 10 µl, 200 µl, 1,000 µl (Sorenson BioScience, catalog numbers: 35210 , 35240 , 35260 ) (see Note 1 for more details)
    7. Qubit® Assay tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: Q32856 )
    8. LightCycler® 480 multiwell plate 96, white (Roche Molecular Systems, catalog number: 04729692001 )
    9. LightCycler® 480 sealing foil (Roche Molecular Systems, catalog number: 04729757001 )
    10. Liquid nitrogen
    11. 20% sodium dodecyl sulfate (SDS) (Biosolve, catalog number: 198123 )
    12. Autoclaved 1 M KCl solution
    13. Autoclaved 5 M NaCl solution
    14. Autoclaved 1 M MgCl2 solution
    15. Sterile filtered 1 M HEPES buffer pH 7.6 (Sigma-Aldrich, catalog number: H0887 )
    16. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
    17. 1 M DL-Dithiothreitol (DTT) prepared from powder (Sigma-Aldrich, catalog number: D0632 ) dissolved in deionized water
    18. 1 M sodium butyrate prepared from powder (Sigma-Aldrich, catalog number: 303410 ) dissolved in deionized water
    19. EDTA-free protease inhibitor cocktail tablets (Roche Molecular Systems, catalog number: 04693132001 )
    20. 16% formaldehyde solution, methanol-free (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 28906 )
    21. 2.5 M glycine prepared from powder (Sigma-Aldrich, catalog number: G8898 ) dissolved in deionized water filtered with a disposable syringe filter (0.45 μm) (Pall, catalog number: 4422 )
    22. 0.5 M EDTA pH 8.0 (Sigma-Aldrich, catalog number: 03690 )
    23. 0.5 M EGTA pH 8.0 prepared from powder (Sigma-Aldrich, catalog number: E3889 ) dissolved in deionized water
    24. 1 M sodium deoxycholate prepared from powder (Sigma-Aldrich, catalog number: D5670 ) dissolved in deionized water
    25. 20% N-Lauroylsarcosine sodium salt solution (Sigma-Aldrich, catalog number: L7414 )
    26. 10 mg/ml RNase A (Sigma-Aldrich, catalog number: R6513 ) dissolved in deionized water
    27. UltraPureTM phenol:chloroform:isoamyl alcohol (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15593049 ) referred as phenol-chloroform in the text
    28. 3 M sodium acetate, pH 5.5 (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9740 )
    29. 35 mg/ml glycogen (MP-Biomedicals, catalog number: 11GLYCO001 )
    30. Ethanol absolute AnalaR NORMAPUR® (VWR, catalog number: 20821.310 )
    31. Agarose powder (Sigma-Aldrich, catalog number: A9539 )
    32. 1x TAE buffer prepared from 50x solution (Biosolve, catalog number: 205023 )
    33. Autoclaved Tris HCl pH 8.0
    34. Ethidium bromide (Sigma-Aldrich, catalog number: E1510 )
    35. Nuclease free water, not DEPC treated (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9937 )
    36. Urea (MP Biomedicals, catalog number: 821519 )
    37. Sucrose (Sigma-Aldrich, catalog number: S0389 )
    38. Bromophenol blue (Sigma-Aldrich, catalog number: B5525 )
    39. 100 bp DNA ladder (New England Biolabs, catalog number: N3231S )
    40. Qubit® dsDNA HS Assay Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q32854 )
    41. Dynabeads® Protein A for Immunoprecipitation (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10001D ) or Dynabeads® Protein G for Immunoprecipitation (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10003D ) (see Note 2 for details)
    42. 20 mg/ml proteinase K (Sigma-Aldrich, catalog number: P2308 )
    43. 1 g/µl of normal rabbit IgG (Cell Signaling Technology, catalog number: 2729 )
    44. Sterile filtered water (Sigma-Aldrich, catalog number: W3500 )
    45. LightCycler® 480 SYBR Green I Master (Roche Molecular Systems, catalog number: 04707516001 )
    46. 10x embryo buffer stock solution (see Recipes)
    47. Embryo buffer (see Recipes)
    48. Buffer A1 (see Recipes)
    49. Lysis buffer 1 (see Recipes)
    50. Lysis buffer 2 (see Recipes)
    51. Lysis buffer wash (see Recipes)
    52. TE buffer
    53. Low blue loading buffer (see Recipes)
    54. Elution buffer 1 (see Recipes)
    55. Elution buffer 2 (see Recipes)

Equipment

  1. Sieves (pores < 0.5 mm)
  2. Microwave
  3. 1 L beaker (Corning, PYREX®, catalog number: 1003-1L )
  4. Magnetic stirrer
  5. Cages (Dutscher Scientific, catalog number: 789092 )
  6. Wash bottle
  7. Paintbrush (Carolina Biological Supply, catalog number: 173094 )
  8. Calibration checked pipettes 0-2 µl, 1-10 µl, 20-200 µl, 100-1,000 µl
  9. Rotating wheels, one in a cold room and the other one at room temperature
  10. Stereomicroscope
  11. Fine #55 forceps for dissection (Fine Science Tools, catalog number: 11255-20 )
  12. WheatonTM Tenbroeck tissue grinder (WHEATON, catalog number: 357422 )
  13. Agarose gel caster
  14. Shaker (For instance the Orbital Shaker from Starlab group) (STARLAB INTERNATIONAL, model: OrbitalTM Shaker, catalog number: N2400-8030 )
  15. Cold centrifuge (4 °C, able to reach at least 14,000 x g)
  16. Cold sonicator (we recommend the Bioruptor® from Diagenode, model: Bioruptor® Plus sonication device , catalog number: B01020001)
  17. Magnetic rack (Thermo Fisher Scientific, model: DynaMagTM-2 Magnet, catalog number: 12321D )
  18. Gel visualization system (UV lamp)
  19. Qubit® fluorometer (Thermo Fisher Scientific, InvitrogenTM, model: Qubit® 3.0 , catalog number: Q33216)
  20. LightCycler® Instrument II (Roche Molecular Systems, model: LightCycler® 480 Instrument II , catalog number: 05015278001)

Procedure

  1. Collection of biological samples
    Note: This step differs for embryos and imaginal discs.
    EMBRYOS:
    1. Melt egg laying medium using a microwave (see Recipes).
    2. Fill the Petri dishes with 15 ml of egg laying medium.
    3. While egg laying medium is cooling down, fill a beaker with 100 ml of sink water and put it on a magnetic stirrer.
    4. Add progressively 50 g of yeast to the beaker. Achieve the mixing by hand stirring as the yeast mixture becomes very viscous.
    5. Once the egg laying medium gets solid, spread 5-10 ml of yeast mixture on each dish (see Figure 1).


      Figure 1. Egg laying medium covered with yeast mixture

    6. Assemble the Petri dishes onto the cages (see Figure 2) and let flies lay eggs as long as required to get a sufficient amount of embryos (see Figure 3).
      Note: See Note 3 to know the number of embryos required.


      Figure 2. Petri dish assembled onto the bottom of a cage containing adult flies


      Figure 3. Petri dish layered with Drosophila embryos (O/N egg laying)

    7. Collect the embryos in a sieve by spraying 1x embryo buffer with a wash bottle (see Figure 4). Use a paintbrush to help.


      Figure 4. Embryos in a sieve (O/N collection)

    8. Wash the embryos three times by putting the sieve in a bath of embryo buffer (use the cap of a tip box).
    9. Collect the embryos with a cut 200 µl tip and transfer them in an Eppendorf tube.
    10. Remove as much buffer as possible and store them at -80 °C.

      IMAGINAL DISCS:

      1. Dissect imaginal discs in Schneider’s medium (SM) (Spratford and Kumar, 2014), and put them in a 1.5 ml LoBind Eppendorf tube, kept on ice and containing 1 ml of SM.
        Note: See Note 4 to know the number of discs required.
      2. Dissection time should not exceed 1 h.
      3. Centrifuge for 2 min (4 °C, 4,000 x g) and remove the supernatant. Imaginal discs can be snap frozen in liquid nitrogen and stored at -80 °C for months, allowing multiple rounds of dissection in order to collect enough material.

  2. Cross-linking proteins to DNA
    Note: At this step, embryos and imaginal discs samples are processed the same way.
    1. If samples have been frozen, thaw them on ice for a couple of minutes.
    2. Resuspend the tissues and pool them in a final volume of 888 µl of ice-cold A1 buffer.
    3. Transfer them into a 2 ml tight Tenbroeck at room temperature (RT).
    4. Fixation: quickly add 112 µl of 16% formaldehyde (FA) in the Tenbroeck (final concentration of 1.8% FA). Fixation is performed at RT.
      Note: See Note 5 for the concentration of FA to be used.
    5. Disrupt the tissues for 5 min by slowly moving the pestle of the Tenbroeck up and down in the mortar.
    6. Transfer the disrupted tissues back to a 1.5 ml LoBind Eppendorf tube.
    7. Put on a rotating wheel at RT: total time for fixation is 15 min.
    8. Stop crosslinking: add 160 µl of 2.5 M glycine (350 mM final) and put on a rotating wheel for 5 min, then put on ice.

      Note: All the following steps are done on ice or at 4 °C unless otherwise specified.

    9. Centrifuge for 2 min (4 °C, at 2,000 x g) and discard the supernatant.
    10. Wash with 1 ml of buffer A1 (disrupt the pellet by pipetting).
    11. Centrifuge for 2 min (4 °C, 2,000 x g).
    12. Repeat steps B9 and B10 twice.
    13. Wash once with lysis buffer 1, centrifuge for 2 min (4 °C, 2,000 x g) and remove the supernatant.

  3. Chromatin isolation and fragmentation by sonication
    Note: This step differs for embryos and imaginal discs samples.
    EMBRYOS:
    1. Prepare 2 ml of 0.1% SDS lysis buffer 2 (10 µl of 20% SDS + 1,990 µl of lysis buffer 2).
    2. Resuspend the pellet with (Number of embryos/1,000) x 250 µl of freshly prepared 0.1% SDS lysis buffer 2.
    3. Incubate for 2 h on ice, and put the ice bucket with the samples in a shaker (600 rpm) to avoid SDS precipitation.
      Note: See Note 6 for an alternative method.
    4. Sonication: split lysate in several 15 ml polystyrene Falcon tubes in order to get 1.5 ml of lysate per tube.
      Note: See Note 7 for considerations about sonication efficiency.
    5. Set the Bioruptor sonicator as follows: Number of cycles = 12, power = HIGH, ON = 30 sec, OFF = 30 sec.
    6. After sonication, pool all the tubes back together. At this step, samples can be snap frozen in liquid nitrogen and conserved at -80 °C for months.

    IMAGINAL DISCS:
    1. Prepare 1 ml of 1% SDS lysis buffer 2 (50 µl of 20% SDS + 950 µl of lysis buffer 2).
    2. Resuspend the pellet with (Number of imaginal discs/600) x 150 µl of freshly prepared 1% SDS lysis buffer 2.
    3. Incubate for 2 h on ice, and put the ice bucket with the samples in a shaker (600 rpm) to avoid SDS precipitation.
      Note: See Note 6 for an alternative method.
    4. Sonication: split lysate in several 1.5 ml LoBind tubes in order to get 150 µl of lysate per tube.
      Note: See Note 7 for considerations about sonication efficiency.
    5. Set the Bioruptor sonicator as follows: Number of cycles = 18, power = HIGH, ON = 30 sec, OFF = 30 sec.
    6. After sonication, pool all the tubes back together. At this step, samples can be snap frozen in liquid nitrogen and conserved at -80 °C for months.

  4. Sonication test
    Note: At this step, embryos and imaginal discs samples are processed the same way. If your sonication conditions are well established, you can skip this step.
    1. For the sonication test, transfer 5-10 µl of the lysate (200 ng of DNA is sufficient) in a fresh 1.5 ml Lobind tube. During the sonication test, keep the remaining lysate at 4 °C.
    2. Bring the volume up to 100 µl with lysis buffer 1.
    3. Add 2 µl of 10 mg/ml RNase A and incubate for 6 h (to O/N) at 65 °C with 450 rpm agitation.
    4. Add 100 µl of phenol-chloroform and vortex for 30 sec.
    5. Centrifuge for 10 min (RT, maximum speed).
    6. Transfer the aqueous phase to a fresh 1.5 ml Lobind tube and add 10 µl of 3 M NaAc (pH 5.5), 2 µl of 35 mg/ml glycogen and 250 µl of 100% EtOH. Vortex and incubate for 30 min at -20 °C.
    7. Centrifuge for 20 min (4 °C, maximum speed).
    8. During centrifugation, prepare a 1.5% agarose gel by dissolving 0.75 g of agarose powder with 50 ml of 1x TAE buffer.
    9. Melt the agarose solution in a microwave, and let it cool down for 5 min before adding 3 µl of ethidium bromide.
    10. Cast the gel in an agarose gel caster and let it cool down at RT.
    11. Once the 20 min centrifugation is over, remove the supernatant and air dry the pellet for 10 min at RT.
    12. Resuspend the pellet in 8 µl of nuclease free H2O and add 2 µl of low blue loading buffer (see Recipes for details).
    13. Deposit 5 µl of 100 bp DNA ladder and the 10 µl of the sonication test on the gel.
    14. Run the gel for 20-25 min at 135 V and observe it using a UV lamp. An example of efficient sonication is depicted in Figure 5.
      Note: See Note 8 for considerations about the importance of sonication quality.


      Figure 5. Ideal sonication gel. 1. 100 bp ladder; 2. Sonication test. The size of bulk DNA fragments ranges from 100 to 300 bp.

  5. Immunoprecipitation
    Note: This step differs for embryos and imaginal discs samples.
    EMBRYOS:
    1. If the sonication pattern is adequate, centrifuge the sonicated lysate for 5 min (4 °C, 14,000 x g). If sonication is insufficient, sonicate the chromatin for 2 cycles more with the same settings and repeat the sonication test.
    2. Transfer the supernatant into a fresh tube (if possible LoBind).
    3. Quantify the DNA using the Qubit® dsDNA HS Assay Kit, following manufacturer’s instructions (1 µl of sonicated lysate should be enough).
      Note: See Note 9 for recommendations about the amount of DNA to be used for IPs.
    4. Split chromatin samples into N aliquots, each containing 250 µl. N = number of IPs + 2 (One Mock and one INPUT).
    5. Wash (number of samples + 2) x 15 µl of Protein A or G Dynabeads.
      Note: See Note 10 to know how to wash the beads and see Note 2 to know the type of Dynabeads to be used (A or G).
    6. Preclearing: Add 15 µl of washed Protein A or G Dynabeads to the chromatin samples and preclear chromatin for 2 h (to O/N) at 4 °C on a rotating wheel.
    7. Put the tubes in the magnetic rack and transfer the precleared chromatin (supernatant) in fresh 1.5 ml LoBind tubes.
    8. Store the tube(s) containing the Input at 4 °C until step F13 (DNA recovery).
    9. Immunoprecipitation: Add the antibodies in the IP tubes and the Mock; incubate for 4 h at 4 °C on a rotating wheel. For the Mock, use the same amount of normal IgG from the same species that has been used for the IPs.
      Note: See Material and Reagents for the catalog number of the rabbit normal IgG we use. See Notes for details about the quantity of antibody to use (Note 11) and which controls can be performed (Notes 12, 13 and 14).
    10. During incubation: wash (number of IPs + 1 Mock) x 30 µl of Protein A or G Dynabeads.
      Note: See Note 10 to know how to wash the beads and see Note 2 to know the type of Dynabeads to be used (A or G).
    11. Add 30 µl of Protein A Dynabeads per sample and incubate O/N at 4 °C on a rotating wheel.

    IMAGINAL DISCS:
    1. If the sonication pattern is adequate, centrifuge the sonicated lysate for 5 min (4 °C, 14,000 x g). If sonication is insufficient, sonicate the chromatin for 5 cycles more with the same settings and repeat the sonication test.
    2. Transfer the supernatant into a fresh 1.5 ml LoBind tube.
    3. Quantify the DNA using the Qubit® dsDNA HS Assay Kit, following manufacturer’s instructions (1 µl of sonicated lysate should be enough).
      Note: See Note 9 for recommendations about the amount of DNA to be used for IPs.
    4. Split chromatin samples into N aliquots, each containing 100 µl. N = number of IPs + 2 (One Mock and one INPUT).
    5. Dilute 10 times by adding 900 µl of lysis buffer 2 (without SDS) in order to reach a 0.1% SDS final concentration. Final volume is 1 ml.
    6. Wash (number of samples + 2) x 15 µl of Protein A or G Dynabeads.
      Note: See Note 10 to know how to wash the beads and see Note 2 to know the type of Dynabeads to be used (A or G).
    7. Preclearing: Add 15 µl of washed Protein A or G Dynabeads to the chromatin samples and preclear chromatin for 2 h (to O/N) at 4 °C on a rotating wheel.
    8. Put the tubes in the magnetic rack and transfer the precleared chromatin (supernatant) in fresh 1.5 ml LoBind tubes.
    9. Store the tube(s) containing the Input at 4 °C until step F13 (DNA recovery).
    10. Immunoprecipitation: Add the antibodies in the IP tubes and the Mock; incubate for 4 h at 4 °C on a rotating wheel. For the Mock, use the same amount of normal IgG from the same species that has been used for the IPs.
      Note: See Materials and Reagents for the catalog number of the rabbit normal IgG we use. See Notes for details about the quantity of antibody to use (Note 11) and which controls can be performed (Notes 12, 13 and 14).
    11. During incubation: wash (number of IPs + 1 Mock) x 30 µl of Protein A or G Dynabeads.
      Note: See Note 10 to know how to wash the beads and see Note 2 to know the type of Dynabeads to be used (A or G).
    12. Add 30 µl of Protein A Dynabeads per sample and incubate O/N at 4 °C on a rotating wheel.

  6. DNA recovery
    Note: At this step, embryos and imaginal discs samples are processed in the same way.
    1. The next day, put the tubes on the magnetic rack and discard the supernatant.
    2. Put 1 ml of lysis buffer wash (kept on ice) in the tubes containing the IPs and the Mock, remove them from the magnetic rack and put them back on ice.
    3. Homogenize by pipetting and incubate for 5 min at 4 °C on a rotating wheel.
    4. Put the tubes on a magnetic rack and remove the supernatant.
    5. Repeat steps F2-F4 three times with lysis buffer wash (4 washes in total) and two times with TE buffer (kept on ice).
    6. After the last wash, be careful to remove all the remaining TE buffer.
    7. First elution: Add 100 µl of elution buffer 1 and homogenize by pipetting.
    8. Incubate the IPs and the Mock at 65 °C for 15 min under vigorous shaking (1,300 rpm).
    9. Put the samples on the magnetic rack and transfer the supernatants in fresh 1.5 ml LoBind tubes.
    10. Second elution: Add 150 µl of elution buffer 2 to the beads and homogenize by pipetting.
    11. Incubate at 65 °C for 15 min under vigorous shaking (1,300 rpm).
    12. Put the tubes on a magnetic rack and pool the two eluates (final volume 250 µl).
    13. Decrosslinking: incubate all the samples for 6 h (to O/N) at 65 °C, 300 rpm. Don’t forget to also take the Input(s) that was/were kept at 4 °C.
    14. The day after, add 3 µl of 20 mg/ml Proteinase K per tube and incubate for 2 h at 56 °C.
    15. Add 1 volume of phenol-chloroform and vortex for 30 sec.
    16. Centrifuge for 15 min (RT, maximum speed).
    17. Transfer aqueous phase to fresh 1.5 ml LoBind tubes and add 1/10 volume of 3 M NaAc, pH 5.5 (25 µl), 2 µl of 35 mg/ml glycogen and 2.5 volumes of 100% EtOH (625 µl).
    18. Precipitation: vortex the samples and incubate them O/N at -20 °C.

  7. Identification of factor associated DNA sequences by PCR or sequencing
    Note: At this step, embryos and imaginal discs samples are processed in the same way.
    1. The day after, centrifuge for 30 min at full speed at 4 °C.
    2. Discard supernatant and wash the pellet with 500 µl of ice-cold 75% EtOH.
    3. Centrifuge for 10 min (4 °C, maximum speed).
    4. Discard supernatant and air dry the pellet for 10 min.
    5. Resuspend the pellet in the required volume of nuclease free H2O (depending on the downstream applications, see Note 15 for more details) and put the tubes at 37 °C for 10 min, 450 rpm.
    6. For ChIP-Seq, see Note 16.
    7. For qPCR, we use 5 µl of Master Mix, 0.1 µl of each primer (50 pmol/µl), 2.8 µl of water and 2 µl of DNA sample.
    8. Before deposition on the PCR plate, the Input is diluted 10 times to keep the same range of Crossing point (Cp) with the one of the IPs.
    9. See Data analysis section to know how to analyze the results.

Data analysis

  1. It is very important to carefully  select the target regions of the oligo pairs. We recommend to use at least two negative controls: one locus that is known to not be enriched for the studied mark/protein, and another one located about 1-5 kb upstream or downstream of the region of interest. A positive control region is also required. Ideally, this region is known to be enriched for the studied mark/protein.
  2. Perform at least two technical duplicates for each oligo pairs.
  3. Once Crossing points (Cps) have been acquired, calculate the mean between the two technical replicates (see Figure 6A):
    MEAN = 2^-(mean(Cp1,Cp2)) to go back to the linear scale.
  4. For each target region, calculate the % of Input for the IP and the MOCK (see Figure 6A):
    % of Input IP= 2^-(MEAN IP)/2^-(MEAN INPUT/10)
    % of Input MOCK= 2^-(MEAN MOCK)/2^-(MEAN INPUT/10)


    Figure 6. PSC ChlP from Eye-Antennal imaginal discs. A. Excel sheet showing the different calculation steps (columns) for replicate 1; B. Histogram showing the relative enrichment of each region expressed in % of Input. The different target regions are represented on the x-axis, and the y-axis corresponds to the % of Input. ‘pgrp body’ is used as a negative control region and is located in the body of the PGRP-LE gene. Antp (Antennapedia) is a well characterized PSC target. dan (tested region) is significantly enriched compared to the negative control. dan is also significantly enriched compared to a region located 1 kb downstream (dan + 1 kb), showing that regions sharing the same higher-order chromatin environment can have different local composition, and providing a further and ideal control.

  5. Results can finally be displayed on a bar plot (see Figure 6B).
  6. Figure 6B shows a qChIP experiment that was performed in triplicate from eye-antennal imaginal discs (500 discs per IP). The PSC antibody was described in (Strutt and Paro, 1997) and 10 µl was used per IP. The following PCR primers were used for qPCR:
    pgrp body: F:cctggtgaatgatagcttactctg
    R:cttactcaaaaccgaagagatcg
    AntpF:tggccgagtttatatcgaagcg
    R:cggccaacttgtgttgttgttc
    danF:cattgtacatttggctgtcacc
    R:gtcagttggtctgccttggtat
    dan + 1 kb: F:ctctctagcttgacgcaccttt
    R:aaactgtttagacccgacatgg
  7. The Mock corresponds to the background level and should therefore be consistently lower than the IP. The enriched regions are the ones that show an enrichment compared to the negative control regions, with no enrichment in the Mock.

Notes

  1. Sterile, DNase and RNase free, DNA low binding filter tips have to be used when the tip gets in contact with the solubilized chromatin. Otherwise, use sterile filter tips.
  2. Protein A Dynabeads are more suitable to work with rabbit IgG while Protein G is preferentially used for mouse IgG.
  3. For 16-18 h old embryos, use at least 500 adult flies and let them lay eggs for 2 h. Remove the adults from the cage and let the embryos develop for 16 h. 1,000 embryos (roughly 50 mg wet weight) are sufficient per IP (histone marks and/or proteins).
  4. For proteins, 400 imaginal discs are required per IP (5 µg of DNA after lysis). For histone marks, 200 are sufficient.
  5. This protocol works with 1.8% FA fixation for proteins. Whereas, for histone marks 1% FA is usually sufficient.
  6. As an alternative to the use of a shaker for the lysis step, it is possible to manually pipette the lysate every 20/30 min to avoid SDS precipitation.
  7. For sonication, 4 parameters are essential: 1) the temperature (the bath has to be maintained at 4 °C), 2) the % of FA used for fixation (the higher the concentration, the longer the sonication time), 3) the volume in which sonication is performed (with the Diagenode Bioruptor, we recommend to not sonicate more than 150 µl of lysate in Eppendorf tubes [imaginal discs] and 1.5 ml in 15 ml polystyrene Falcon [embryos]), 4) the concentration of SDS in the lysis buffer (more SDS means easier sonication).
  8. The quality of sonication is crucial, since it will determine the resolution of the ChIP experiment. A good average size is 200-300 bp.
  9. Use 2-5 µg of DNA for histone marks and 5-10 µg for proteins. Nevertheless, the quality of the IP also depends highly on the quality and the quantity of antibody used.
  10. Dynabeads washes:
    1. Before taking the beads, resuspend them carefully.
    2. Put the tube containing the beads on a magnetic rack and remove the buffer.
    3. Add 3 times the volume of lysis buffer 2 (as an example, for 75 µl of beads wash with 3 x 75 = 225 µl of lysis buffer 2), take the tubes out of the rack and homogenize by pipetting to rinse the beads.
    4. Put the tubes containing the beads on a magnetic rack, discard the supernatant and repeat the previous step.
    5. Put the tubes containing the beads on a magnetic rack and discard the buffer. Resuspend the beads in the initial volume ([number of samples + 2] x 15 µl or [number of samples + 1] x 30).
  11. We recommend to use 5 µg of antibody per IP, and the same quantity for the Mock.
  12. Mock (non-specific IgG) and Input are good controls to assess the specificity and the enrichment of the protein over the background. For ChIP-Seq, only the Input is mandatory.
  13. Another possible control consists in using a specific antibody for a protein/histone mark that should not colocalize with your protein/histone mark of interest.
  14. Ideally, the specificity of the antibody should be assessed by performing a ChIP in a K.O or K.D tissue.
  15. For ChIP-Seq, resuspend in 25 µl of nuclease free water. For qChIP, resuspend in the required volume to perform your measurements in duplicates.
  16. For ChIP-Seq, use 1 µl of DNA sample to quantify the DNA using Qubit® dsDNA HS Assay Kit. Then, use 2 µl of DNA sample dissolved in as much nuclease free water as necessary to test 3-4 loci by qPCR (don’t forget to take the dilution in account when analyzing the data). If qPCR controls are satisfying, samples can be used for library preparation and sequencing.

Recipes

  1. Egg laying medium (325 ml)
    8.75 g of agar mixed in 275 ml of water are dissolved in a microwave. Then 48 ml of vinegar and 3.2 ml of 10% Moldex are added with 50 mg of neutral red
  2. 10x embryo buffer stock solution
    7% NaCl
    0.5% Triton X-100

    Note: The solutions described below should not be stored for more than 1 week at 4 °C.

  3. Embryo buffer
    Dilute 10 times the 10x stock solution with deionized water
  4. Buffer A1
    60 mM KCl
    15 mM NaCl
    4 mM MgCl2
    15 mM HEPES
    0.5% Triton X-100
    0.5 mM DTT
    10 mM sodium butyrate in sterile filtered water
    Add one tablet of protease inhibitor cocktail each 50 ml
  5. Lysis buffer 1
    140 mM NaCl
    15 mM HEPES
    1 mM EDTA
    0.5 mM EGTA
    1% Triton X-100
    0.5 mM DTT
    0.1% sodium deoxycholate
    10 mM sodium butyrate in sterile filtered water (Sigma-Aldrich)
    Add one tablet of protease inhibitor cocktail each 50 ml
    Note: Lysis buffer 1 will also be used to prepare lysis buffer 2 and lysis buffer wash.
  6. Lysis buffer 2
    0.5% N-lauroylsarcosine in FRESH lysis buffer 1
  7. Lysis buffer wash
    0.05% SDS in FRESH lysis buffer 1
  8. TE buffer
    0.1 mM EDTA
    10 mM Tris HCl pH 8.0 in sterile filtered water (Sigma-Aldrich)
  9. Low blue loading buffer
    4 M urea
    50% sucrose
    1 mM EDTA
    Few grains of bromophenol blue
    Note: The bromophenol blue migrates at the same size than sonicated chromatin, and should therefore be kept as much diluted as possible.
  10. Elution buffer 1
    10 mM EDTA
    50 mM Tris HCl pH 8.0
    1% SDS in sterile filtered water
  11. Elution buffer 2
    0.1 mM EDTA
    10 mM Tris HCl pH 8.0
    0.67% SDS

Acknowledgments

We thank all the authors of the article from which this protocol was adapted from (Loubiere et al., 2016). We thank Frédéric Bienvenu (Institute of Functional Genomics, Montpellier, France) for his technical support. We thank Filippo Ciabrelli for discussions and help in writing this manuscript.

References

  1. Loubiere, V., Delest, A., Thomas, A., Bonev, B., Schuettengruber, B., Sati, S., Martinez, A. M. and Cavalli, G. (2016). Coordinate redeployment of PRC1 proteins suppresses tumor formation during Drosophila development. Nat Genet 48(11): 1436-1442.
  2. Spratford, C. M. and Kumar, J. P. (2014). Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster. J Vis Exp (91): 51792.
  3. Strutt, H. and Paro, R. (1997). The polycomb group protein complex of Drosophila melanogaster has different compositions at different target genes. Mol Cell Biol 17(12): 6773-6783.

简介

与qPCR(qChIP)或高通量测序(ChIP-Seq)相结合的染色质免疫沉淀已被广泛用于识别转录因子的DNA结合位点或基因组中各种组蛋白标记的定位。 ChIP实验通常包括7个步骤:收集生物样品(A),交联蛋白质到DNA(B),染色质分离和通过超声处理分离(C),超声处理测试(D),用针对蛋白质的抗体进行免疫沉淀感兴趣的组蛋白标记(E),DNA回收(E),通过PCR或测序鉴定因子相关DNA序列(F)。这里描述的协议可以容易地用于ChIP-seq和ChIP-qPCR实验。描述在完整的果蝇组织中优化分析的实验设置条件的整个过程可以在四天内完成。

背景 尽管永生化的培养细胞广泛用于研究各种细胞类型的染色质景观,但是在生理条件下在体内探测相互作用的有价值的方法对于进行转录的时间或空间比较分析是必要的因子和组蛋白修饰图在不同阶段的果蝇发展或不同组织之间。在这里,我们提供了一个详细的ChIP协议,已被优化,以便在整个果蝇胚胎和幼虫成像光盘上工作,突出关键的实验参数。

关键字:ChIP, 果蝇, 胚胎, 成虫盘, 表观遗传标记, 转录因子

材料和试剂

  1. 阴影椎间盘切开步骤
    1.微量移液器的无菌提示,1,000μl
    2. 1.5 ml DNA低粘合管(Eppendorf,目录号:022431021)
    施耐德昆虫培养基(Sigma-Aldrich,目录号:S0146)

  2. 胚胎收集步骤
    1. 培养皿
    2. 1.5ml DNA低结合(LoBind)管(Eppendorf,目录号:022431021)
    3. 果蝇胚胎
    4. SAF-INSTANT酵母(Lesaffre)
    5. 液氮
    6. 琼脂
    7. 溶于水的10%Moldex(Methyl-4-羟基苯甲酸酯)(Sigma-Aldrich,目录号:W271004)
    8. 中性红(Sigma-Aldrich,目录号:N4638)
    9. 铺鸡蛋糕(见食谱)

  3. 染色质免疫沉淀方案
    1. 1.5ml DNA低结合(LoBind)管(Eppendorf,目录号:022431021)
    2. 15 ml无菌塑料管(Greiner Bio One International,目录号:188261)
    3. 50ml无菌塑料管(Greiner Bio One International,目录号:227261)
    4. 15ml聚苯乙烯Falcon管(Corning,目录号:352095)
    5. 无菌过滤嘴10μl,200μl,1,000μl(STARLAB INTERNATIONAL,目录号:S1121-2710,S1120-8810,S1122-1830)(详见附注1)
    6. 无菌,DNA酶和RNA酶免费,DNA低结合过滤嘴10μl,200μl,1,000μl(Sorenson BioScience,目录号:35210,35240,35260)(详见附注1)
    7. Qubit ®测定管(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:Q32856)
    8. LightCycler ®480多孔板96,白(Roche Molecular Systems,目录号:04729692001)
    9. LightCycler ® 480密封箔(Roche Molecular Systems,目录号:04729757001)
    10. 液氮
    11. 20%十二烷基硫酸钠(SDS)(Biosolve,目录号:198123)
    12. 高压灭菌1 M KCl溶液
    13. 高压灭菌5 M NaCl溶液
    14. 高压灭菌1 M MgCl 2 溶液
    15. 无菌过滤的1M HEPES缓冲液pH 7.6(Sigma-Aldrich,目录号:H0887)
    16. Triton X-100(Sigma-Aldrich,目录号:T8787)
    17. 将溶于去离子水的粉末(Sigma-Aldrich,目录号:D0632)制备的1 M DL-二硫苏糖醇(DTT)
    18. 1 M由粉末(Sigma-Aldrich,目录号:303410)制成的丁酸钠溶于去离子水中
    19. 不含EDTA的蛋白酶抑制剂混合片剂(Roche Molecular Systems,目录号:04693132001)
    20. 16%甲醛溶液,不含甲醇(Thermo Fisher Scientific,Thermo Scientific TM,目录号:28906)
    21. 将2.5M甘氨酸溶于用一次性注射器过滤器(0.45μm)过滤的去离子水中(粉末(Sigma-Aldrich,目录号:G8898))(Pall,目录号:4422)
    22. 0.5M EDTA pH8.0(Sigma-Aldrich,目录号:03690)
    23. 溶解在去离子水中的粉末(Sigma-Aldrich,目录号:E3889)制备的0.5M EGTA pH8.0
    24. 1 M溶解在去离子水中的由粉末(Sigma-Aldrich,目录号:D5670)制备的脱氧胆酸钠
    25. 20%N-月桂酰肌氨酸钠盐溶液(Sigma-Aldrich,目录号:L7414)
    26. 10毫克/毫升溶解在去离子水中的RNase A(Sigma-Aldrich,目录号:R6513)
    27. UltraPure 苯酚:氯仿:异戊醇(Thermo Fisher Scientific,Invitrogen TM,目录号:15593049)在文本中被称为苯酚 - 氯仿
    28. 3M醋酸钠,pH5.5(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9740)
    29. 35 mg / ml糖原(MP-Biomedicals,目录号:11GLYCO001)
    30. 乙醇绝对AnalaR NORMAPUR ®(VWR,目录号:20821.310)
    31. 琼脂糖粉(Sigma-Aldrich,目录号:A9539)
    32. 从50x溶液制备的1x TAE缓冲液(Biosolve,目录号:205023)
    33. 高压灭菌Tris HCl pH 8.0
    34. 溴化乙锭(Sigma-Aldrich,目录号:E1510)
    35. 无脱氧核糖核酸酶,不经DEPC处理(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9937)
    36. 尿素(MP Biomedicals,目录号:821519)
    37. 蔗糖(Sigma-Aldrich,目录号:S0389)
    38. 溴苯酚蓝(Sigma-Aldrich,目录号:B5525)
    39. 100 bp DNA ladder(New England Biolabs,目录号:N3231S)
    40. Qubit ® dsDNA HS测定试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:Q32854)
    41. Dynabeads ®用于免疫沉淀的蛋白A(Thermo Fisher Scientific,Invitrogen TM,目录号:10001D)或Dynabeads用于免疫沉淀的蛋白G(Thermo Fisher Scientific,Invitrogen TM ,目录号:10003D)(详见附注2)
    42. 20mg / ml蛋白酶K(Sigma-Aldrich,目录号:P2308)
    43. 1μg/μl正常兔IgG(Cell Signaling Technology,目录号:2729)
    44. 无菌过滤水(Sigma-Aldrich,目录号:W3500)
    45. LightCycler ® 480 SYBR Green I Master(Roche Molecular Systems,目录号:04707516001)
    46. 10x胚胎缓冲液储备溶液(参见食谱)
    47. 胚胎缓冲液(参见食谱)
    48. 缓冲液A1(参见食谱)
    49. 裂解缓冲液1(参见食谱)
    50. 裂解缓冲液2(参见食谱)
    51. 裂解缓冲液洗涤(参见食谱)
    52. TE缓冲区
    53. 低蓝色加载缓冲区(见配方)
    54. 洗脱缓冲液1(参见食谱)
    55. 洗脱缓冲液2(参见食谱)

设备

  1. 筛(孔<0.5mm)
  2. 微波
  3. 1升烧杯(Corning,PYREX ®,目录号:1003-1L)
  4. 磁力搅拌器
  5. 笼(Dutscher Scientific,目录号:789092)
  6. 洗瓶
  7. 油漆刷(Carolina Biological Supply,目录号:173094)
  8. 校准检查移液器0-2μl,1-10μl,20-200μl,100-1,000μl
  9. 旋转轮,一个在寒冷的房间,另一个在室温下
  10. 立体显微镜
  11. 精细#55镊子解剖(精细科学工具,目录号:11255-20)
  12. Wheaton TM Tenbroeck组织研磨机(WHEATON,目录号:357422)
  13. 琼脂糖凝胶脚轮
  14. 振荡器(例如来自starlabgroup的轨道振荡器)(STARLAB INTERNATIONAL,型号:Orbital TM 振动器,目录号:N2400-8030)
  15. 冷离心机(4°C,能够达到至少14,000 x g)
  16. 冷超声波仪(我们推荐来自Diagenode的Bioruptor ®型号:Bioruptor ® Plus超声波设备,目录号:B01020001)
  17. 磁性架(Thermo Fisher Scientific,型号:DynaMag TM -2磁铁,目录号:12321D)
  18. 凝胶可视化系统(UV灯)
  19. Qubit ®荧光计(Thermo Fisher Scientific,Invitrogen TM,型号:Qubit 3.0,目录号:Q33216)
  20. LightCycler ®仪器II(Roche Molecular Systems,型号:LightCycler 480仪器II,目录号:05015278001)

程序

  1. 生物样本的收集
    注意:此步骤对于胚胎和图像光盘是不同的。
    胚胎:
    1. 使用微波炉的融合蛋产品介质(见食谱)。
    2. 用培养皿中的15毫升填充培养皿。
    3. 在放置培养基的同时,将100ml的水槽放入烧杯中,放在磁力搅拌器上
    4. 逐渐加入50克酵母到烧杯中。当酵母混合物变得非常粘稠时,通过手动搅拌实现混合
    5. 一旦产卵培养基变得固体,每个盘上撒上5-10ml酵母混合物(见图1)

      图1.用酵母混合物覆盖的铺蛋液

    6. 将培养皿装配到笼子上(见图2),并让苍蝇放置鸡蛋,只要要获得足够数量的胚胎(见图3)。
      注意:见注3,了解所需的胚胎数量。


      图2.组装到包含成年苍蝇的笼子底部的培养皿


      图3.用 果蝇 胚胎(O / N产蛋)分层的培养皿

    7. 通过用洗涤瓶喷洒1x胚胎缓冲液,收集筛子中的胚胎(见图4)。使用画笔来帮助。


      图4.筛子中的胚胎(O / N收集)

    8. 通过将筛子放入胚胎缓冲液浴中(使用提示盒的盖子)将胚胎洗涤三次。
    9. 收集胚胎与切200毫升的尖端并转移他们在一个Eppendorf管。
    10. 删除尽可能多的缓冲区,并将它们存储在-80°C。

      图片:

      1. 在Schneider's培养基(SM)(Spratford和Kumar,2014)中分解成像圆盘,并将它们放入1.5ml LoBind Eppendorf管中,保持在冰上并含有1ml SM。
        注意:请参阅注释4了解所需的光盘数量。
      2. 解剖时间不应超过1小时。
      3. 离心2分钟(4℃,4,000xg),并除去上清液。想象的盘可以在液氮中快速冷冻并储存在-80°C几个月,允许多轮解剖以收集足够的物质。

  2. 将蛋白质与DNA交联
    注意:在此步骤中,胚胎和成像光盘样品以相同的方式处理。
    1. 如果样品已经冻结,在冰上解冻几分钟。
    2. 重新悬挂组织,并将其汇集在最终体积为888μl的冰冷的A1缓冲液中
    3. 将它们在室温(RT)下转移到2ml紧的Tenbroeck中
    4. 固定:在Tenbroeck(终浓度为1.8%FA)中快速加入112μl16%甲醛(FA)。固定在室温下进行。
      注意:有关使用FA浓度的说明,请参见注5。
    5. 通过在灰浆中缓慢地移动Tenbroeck的杵,将组织破碎5分钟。
    6. 将破碎的组织转移回1.5 ml LoBind Eppendorf管。
    7. 在RT上旋转轮:固定时间为15分钟。
    8. 停止交联:加入160μl2.5M甘氨酸(最终350mM),放在旋转轮上5分钟,然后放在冰上。

      注意:除非另有说明,所有以下步骤均在冰面或4°C下进行。

    9. 离心2分钟(4℃,2,000xg),弃去上清液。
    10. 用1ml缓冲液A1洗涤(通过移液来破坏沉淀)。
    11. 离心2分钟(4°C,2,000 x g)
    12. 重复步骤B9和B10两次。
    13. 用裂解缓冲液1洗涤一次,离心2分钟(4℃,2,000×g),并除去上清液。

  3. 通过超声处理染色质分离和碎裂
    注意:这个步骤对于胚胎和想象的光盘样本是不同的。
    胚胎:
    1. 准备2ml 0.1%SDS裂解缓冲液2(10μl20%SDS +1,990μl裂解缓冲液2)。
    2. 用(数量/ 1,000)×250μl新鲜制备的0.1%SDS裂解缓冲液2重悬沉淀物。
    3. 在冰上孵育2小时,并将冰桶与样品放在振荡器(600rpm)中以避免SDS沉淀。
      注意:有关替代方法,请参见注释6。
    4. 超声处理:在几个15ml聚苯乙烯Falcon管中裂解裂解物,以便每管获得1.5ml裂解物。
      注意:有关超声处理效率的注意事项,请参见附注7.
    5. 设置Bioruptor超声波仪器如下:循环次数= 12,功率=高,ON = 30秒,OFF = 30秒。
    6. 超声处理后,将所有管道放回原处。在此步骤中,样品可以在液氮中快速冷冻,并在-80℃保存数月。

    图片:
    1. 准备1ml 1%SDS裂解缓冲液2(50μl20%SDS +950μl裂解缓冲液2)。
    2. 用(数量的圆盘/ 600)×150μl新鲜制备的1%SDS裂解缓冲液2重悬沉淀物。
    3. 在冰上孵育2小时,并将冰桶与样品放在振荡器(600rpm)中以避免SDS沉淀。
      注意:有关替代方法,请参见注释6。
    4. 超声处理:在几个1.5 ml LoBind管中裂解裂解液,以便每管获得150μl裂解物。
      注意:有关超声处理效率的注意事项,请参见附注7.
    5. 设置Bioruptor超声波仪器如下:循环次数= 18,功率=高,ON = 30秒,OFF = 30秒。
    6. 超声处理后,将所有管道放回原处。在此步骤中,样品可以在液氮中快速冷冻,并在-80℃保存数月。

  4. 超声波测试
    注意:在此步骤中,胚胎和成像光盘样品以相同的方式进行处理。如果您的超声条件已经确定,您可以跳过此步骤。
    1. 对于超声处理测试,在新鲜的1.5ml Lobind管中转移5-10μl裂解物(200ng DNA是足够的)。在超声处理试验中,将剩余的裂解物保持在4℃。
    2. 用裂解缓冲液1将体积达100μl。
    3. 加入2μl10 mg / ml核糖核酸酶A,并在65℃下以450rpm搅拌孵育6小时(至O / N)。
    4. 加入100μl酚 - 氯仿,旋涡30秒
    5. 离心10分钟(RT,最大速度)。
    6. 将水相转移到新鲜的1.5ml Lobind管中,加入10μl3M NaAc(pH 5.5),2μl35mg / ml糖原和250μl100%EtOH。涡旋并在-20℃下孵育30分钟。
    7. 离心20分钟(4℃,最大速度)
    8. 在离心过程中,通过用0.75毫升1×TAE缓冲液溶解0.75克琼脂糖粉末制备1.5%琼脂糖凝胶。
    9. 将琼脂糖溶液在微波炉中熔化,然后冷却5分钟,然后加入3μl溴化乙锭。
    10. 将凝胶投入琼脂糖凝胶浇铸机,让其在室温下冷却。
    11. 一旦离心20分钟,取出上清液,然后将其在室温下空气干燥10分钟
    12. 将沉淀重悬于8μl无核酸酶的H 2 O 2中,加入2μl低蓝色加载缓冲液(详见配方)。
    13. 在凝胶上沉积5μl100 bp DNA梯和10μl超声波测试。
    14. 在135 V下运行凝胶20-25分钟,并使用紫外灯观察。高效超声处理的一个例子如图5所示 注意:关于超声处理质量的重要性的注意事项,请参见注8。


      图5.理想的超声波凝胶。 1。 100bp梯;超声波测试体积DNA片段的大小范围为100〜300bp。

  5. 免疫沉淀
    注意:这个步骤对于胚胎和想象的光盘样本是不同的。
    胚胎:
    1. 如果超声处理模式是足够的,将超声处理的裂解物离心5分钟(4℃,14,000×10 7 g)。如果超声处理不足,请以相同的设置超声染色质2个循环,重复超声处理。
    2. 将上清液转移到新鲜管中(如果可能的话)将其转移到新管中
    3. 使用Qubit dsDNA HS测定试剂盒按照制造商的说明书(1μl超声处理的裂解液应足够)定量DNA。
      注意:请参阅注9,了解有关IP的IP数量的建议。
    4. 将染色质样品分成N份,每份含有250μl。 N = IP数量+ 2(一个模拟器和一个输入)。
    5. 洗涤(样品数量+ 2)×15μl蛋白A或G Dynabeads。
      注意:请参见注10,了解如何清洗珠子,并参见注2,了解要使用的Dynabeads的类型(A或G)。
    6. 预清洗:将15μl洗涤的蛋白A或G Dynabeads加入到染色质样品中,并在4°C的旋转轮上预清洁染色质2小时(至<0> O / N ) 。
    7. 将管放在磁性架中,并将预清除的染色质(上清液)转移到新鲜的1.5 ml LoBind管中。
    8. 将含有输入管的管保存在4℃,直到步骤F13(DNA回收)。
    9. 免疫沉淀:将抗体添加到IP管和Mock中;在4°C的旋转轮上孵育4 h。对于Mock,使用与用于知识产权的相同物种相同数量的正常IgG。
      注意:有关我们使用的兔正常IgG的目录号,请参阅材料和试剂。有关要使用的抗体数量(注11)以及可以执行哪些控件的详细信息,请参阅注释(注12,13和14)。
    10. 孵育期间:洗涤(IPs + 1模拟物)×30μl蛋白A或G Dynabeads。
      注意:请参见注10,了解如何清洗珠子,并参见注2,了解要使用的Dynabeads的类型(A或G)。
    11. 每个样品加入30μl蛋白A Dynabeads,并在4°C的旋转轮上孵育 O / N

    图片:
    1. 如果超声处理模式是足够的,将超声处理的裂解物离心5分钟(4℃,14,000×g)。如果超声处理不足,请以相同的设置将染色质超声5次,并重复超声处理。
    2. 将上清液转移到新鲜的1.5 ml LoBind管中
    3. 使用Qubit dsDNA HS测定试剂盒按照制造商的说明书(1μl超声处理的裂解液应足够)定量DNA。
      注意:请参阅注9,了解有关IP的IP数量的建议。
    4. 将染色质样品分成N等分试样,每个样品含有100μl。 N = IP数量+ 2(一个模拟器和一个输入)。
    5. 通过加入900μl裂解缓冲液2(不含SDS)稀释10倍,以达到0.1%SDS终浓度。最终体积为1 ml。
    6. 洗涤(样品数量+ 2)×15μl蛋白A或G Dynabeads。
      注意:请参见注10,了解如何清洗珠子,并参见注2,了解要使用的Dynabeads的类型(A或G)。
    7. 预清洗:将15μl洗涤的蛋白A或G Dynabeads加入到染色质样品中,并在4°C的旋转轮上预清洁染色质2小时(至<0> O / N ) 。
    8. 将管放在磁性架中,并将预清除的染色质(上清液)转移到新鲜的1.5 ml LoBind管中。
    9. 将含有输入管的管保存在4℃,直到步骤F13(DNA回收)。
    10. 免疫沉淀:将抗体添加到IP管和Mock中;在4°C的旋转轮上孵育4 h。对于Mock,使用与用于知识产权的相同物种相同数量的正常IgG。
      注意:有关我们使用的兔正常IgG的目录号,请参阅材料和试剂。有关要使用的抗体数量(注11)以及可以执行哪些控件的详细信息,请参阅注释(注12,13和14)。
    11. 孵育期间:洗涤(IPs + 1模拟物)×30μl蛋白A或G Dynabeads。
      注意:请参见注10,了解如何清洗珠子,并参见注2,了解要使用的Dynabeads的类型(A或G)。
    12. 每个样品加入30μl蛋白A Dynabeads,并在4°C的旋转轮上孵育 O / N

  6. DNA恢复
    注意:在此步骤中,以相同的方式处理胚胎和成像光盘样本。
    1. 第二天将管放在磁性架上,弃上清
    2. 在含有IP和Mock的管中放入1ml裂解缓冲液洗涤(保存在冰上),将其从磁性架上取出并将其放回冰上。
    3. 通过移液均质,并在4°C的旋转轮上孵育5分钟。
    4. 将管放在磁性架上,取出上清液。
    5. 用裂解缓冲液洗涤重复步骤F2-F4(总共4次洗涤),用TE缓冲液(保存在冰上)两次。
    6. 最后一次洗涤后,小心删除所有剩余的TE缓冲液。
    7. 第一次洗脱:加入100μl洗脱缓冲液1,用移液管匀浆
    8. 在剧烈摇晃(1,300rpm)下,将IP和模拟物在65℃下孵育15分钟。
    9. 将样品放在磁性架上,并将上清液转移到新鲜的1.5 ml LoBind管中
    10. 第二次洗脱:将150μl洗脱缓冲液2加入珠中并通过移液使其均质。
    11. 在剧烈振荡(1,300rpm)下,在65℃下孵育15分钟
    12. 将管放在磁性架上,并将两个洗脱液合并(最终体积250μl)
    13. 去交联:在65°C,300 rpm下孵育所有样品6 h(至 O / N )。不要忘记也采取在4°C保存的输入。
    14. 第二天,每管加入3μl20 mg / ml蛋白酶K,并在56°C孵育2 h。
    15. 加入1体积的苯酚 - 氯仿并涡旋30秒
    16. 离心15分钟(RT,最大速度)。
    17. 将水相转移到新鲜的1.5ml LoBind管中,并加入1/10体积的3M NaAc,pH 5.5(25μl),2μl35mg / ml糖原和2.5体积的100%EtOH(625μl)。 >
    18. 降水:使样品涡旋,并在-20°C下孵育它们的O / N

  7. 通过PCR或测序鉴定因子相关DNA序列
    注意:在此步骤中,以相同的方式处理胚胎和成像光盘样本。
    1. 第二天,在4℃全速离心30分钟。
    2. 弃去上清液,并用500μl冰冷的75%EtOH洗涤沉淀。
    3. 离心10分钟(4℃,最大速度)。
    4. 弃上清,空气干燥沉淀10分钟
    5. 将沉淀物悬浮在所需体积的无核酸酶H 2 O(取决于下游应用,详见附注15),并将管置于37℃10分钟,450rpm。 br />
    6. 对于ChIP-Seq,请参见注释16.
    7. 对于qPCR,我们使用5μl的Master Mix,0.1μl的各引物(50pmol /μl),2.8μl的水和2μl的DNA样品。
    8. 在PCR板上沉积之前,输入“稀释10倍”,以保持与其中一个IP的交叉点(Cp)相同的范围。
    9. 请参阅数据分析部分了解如何分析结果。

数据分析

  1. 仔细选择oligo对的目标区域是非常重要的。我们建议使用至少两个阴性对照:一个已知不被研究的标记/蛋白质富集的位点,另一个位于感兴趣区域上游或下游约1-5kb的位点。还需要阳性对照区域。理想情况下,已知该区域被浓缩用于研究的标记/蛋白质
  2. 对每个寡核苷酸对执行至少两个技术重复。
  3. 一旦获得了交叉点(Cps),计算两次技术重复之间的平均值(见图6A):
    MEAN = 2 ^ - (平均值(Cp1,Cp2))返回线性刻度。
  4. 对于每个目标区域,计算 IP 和 MOCK 的输入的百分比(见图6A):
    输入IP的百分比= 2 ^ - ( MEAN IP )/ 2 ^ - ( MEAN INPUT / 10
    输入MOCK的%= 2 ^ - ( MEAN MOCK )/ 2 ^ - ( MEAN INPUT / 10


    图6.来自Eye-Antennal想象光盘的PSC ChlP。 A.显示复制1的不同计算步骤(列)的Excel表格; B.显示每个区域相对浓度的直方图,以百分比表示。不同的目标区域在x轴上表示,y轴对应于Input的%。 ' pgrp body'用作阴性对照区,位于pGRP-LE 基因的体内。 Antp(Antennapedia)是一个表征良好的PSC目标。与阴性对照组相比,dan (测试区域)显着丰富。与位于1kb下游( + 1kb)的区域相比,eman dan 也显着丰富,表明共享相同高阶染色质环境的区域可以具有不同的局部组成,并提供进一步和理想的控制。

  5. 结果可以最终显示在条形图上(见图6B)。
  6. 图6B示出了从眼 - 触角成像盘(每个IP 500个光盘)一式三份进行的qChIP实验。在(Strutt和Paro,1997)中描述了PSC抗体,并且每IP使用10μl。以下PCR引物用于qPCR:
    pgrp 正文:&nbsp; → F:cctggtgaatgatagcttactctg
    R:cttactcaaaaccgaagagatcg
    Antp :&nbsp; → F:tggccgagtttatatcgaagcg
    R:cggccaacttgtgttgttgttc
    dan :&nbsp; → F:cattgtacatttggctgtcacc
    R:gtcagttggtctgccttggtat
    dan + 1 kb:&nbsp; → F:ctctctagcttgacgcaccttt
    R:aaactgtttagacccgacatgg
  7. 模拟器对应于背景级别,因此应始终低于IP。丰富的地区是与阴性对照区相比富集的地区,而不是在模拟中富集。

笔记

  1. 无菌,DNA酶和RNA酶游离,当尖端与溶解的染色质接触时,必须使用DNA低结合过滤嘴。否则,使用无菌过滤嘴。
  2. 蛋白A Dynabeads更适用于兔IgG,而蛋白G优先用于小鼠IgG。
  3. 对于16-18小时的胚胎,使用至少500只成年苍蝇,让他们下蛋2小时。从笼子中取出成年人,让胚胎发育16 h。每个IP(组蛋白标记和/或蛋白质),1,000个胚胎(约50毫克湿重)足够了
  4. 对于蛋白质,每IP需要400张想象的光盘(裂解后5微克的DNA)。对于组蛋白标记,200是足够的。
  5. 该协议适用于蛋白质的1.8%FA固定。而组蛋白标记1%FA通常就足够了
  6. 作为使用摇床进行裂解步骤的替代方法,可以每20分钟手动移液裂解物以避免SDS沉淀。
  7. 对于超声处理,4个参数是必不可少的:1)温度(浴必须保持在4℃),2)用于固定的FA的%(浓度越高,超声处理时间越长),3)进行超声处理的体积(使用Diagenode Bioruptor,我们建议不要在Eppendorf管[图像圆盘]中超声波超过150μl的裂解液,并在15 ml聚苯乙烯Falcon [胚胎]中超声波清洗),4)SDS的浓度裂解缓冲液(更多的SDS意味着更容易超声处理)。
  8. 超声处理的质量至关重要,因为它将决定ChIP实验的分辨率。平均大小为200-300 bp。
  9. 使用2-5μgDNA作为组蛋白标记,5-10μg用于蛋白质。然而,IP的质量也很大程度上取决于所用抗体的质量和数量
  10. Dynabeads洗涤:
    1. 在服用珠子之前,请仔细重新悬挂
    2. 将含有珠子的管放在磁性架上并取出缓冲液。
    3. 加入裂解缓冲液2的体积的3倍(例如,对于用3×75 =225μl裂解缓冲液2洗涤的75μl珠),将管从架上取出并通过移液冲洗珠进行均质化。 br />
    4. 将含有珠粒的管放在磁性架上,弃去上清液,重复上一步骤。
    5. 将含有珠粒的管放在磁性架上并丢弃缓冲液。将珠子重新悬浮在初始体积([样品数量+ 2]×15μl或[样品数量+ 1]×30)中。
  11. 我们建议每IP使用5微克抗体,同样数量的Mock。
  12. 模拟(非特异性IgG)和输入是评估蛋白质在背景下的特异性和富集的良好对照。对于ChIP-Seq,只有输入是强制性的。
  13. 另一种可能的对照是使用特异性抗体来进行蛋白质/组蛋白标记,该标记不应该与您所感兴趣的蛋白质/组蛋白标记共定位。
  14. 理想情况下,应通过在K.O或K.D组织中进行ChIP评估抗体的特异性
  15. 对于ChIP-Seq,重悬于25μl无核酸酶的水中。对于qChIP,重新悬挂在所需的卷中,以重复的方式执行测量。
  16. 对于ChIP-Seq,使用1μlDNA样品使用Qubit dsDNA HS Assay Kit定量DNA。然后,根据需要使用2μlDNA样品溶解在无需核酸酶的水中,通过qPCR测试3-4个基因座(分析数据时,不要忘记考虑稀释度)。如果qPCR对照是令人满意的,样品可以用于文库的制备和测序。

食谱

  1. 铺鸡蛋(325毫升)
    将8.75g在275ml水中混合的琼脂溶解在微波中。然后加入48ml醋和3.2ml 10%Moldex,加入50mg中性红色
  2. 10x胚胎缓冲液溶液
    7%NaCl
    0.5%Triton X-100

    注意:以下描述的解决方案不应在4°C下储存超过1周。

  3. 胚胎缓冲液
    用去离子水稀释10倍10倍的储备溶液
  4. 缓冲区A1
    60 mM KCl
    15 mM NaCl
    4mM MgCl 2
    15 mM HEPES
    0.5%Triton X-100
    0.5 mM DTT
    10毫克丁酸钠在无菌过滤水中 加入一片蛋白酶抑制剂鸡尾酒每50ml 50ml
  5. 裂解缓冲液1
    140 mM NaCl
    15 mM HEPES
    1 mM EDTA
    0.5 mM EGTA
    1%Triton X-100
    0.5 mM DTT
    0.1%脱氧胆酸钠
    10毫克丁酸钠无菌过滤水(Sigma-Aldrich)
    加入一片蛋白酶抑制剂鸡尾酒每50ml 50ml 注意:裂解缓冲液1也将用于制备裂解缓冲液2和裂解缓冲液洗涤。
  6. 裂解缓冲液2
    FRESH裂解缓冲液1中的0.5%N-月桂酰肌氨酸
  7. 裂解缓冲液洗涤
    在FRESH裂解缓冲液1中的0.05%SDS
  8. TE缓冲区
    0.1 mM EDTA
    无菌过滤水(Sigma-Aldrich)中的10mM Tris HCl pH8.0
  9. 低蓝色加载缓冲区
    4 M尿素
    50%蔗糖
    1 mM EDTA
    少量的溴酚蓝
    注意:溴酚蓝以与超声处理的染色质相同的尺寸迁移,因此应尽可能多地稀释。
  10. 洗脱缓冲液1
    10 mM EDTA
    50 mM Tris HCl pH 8.0
    无菌过滤水中的1%SDS
  11. 洗脱缓冲液2
    0.1 mM EDTA
    10 mM Tris HCl pH 8.0
    0.67%SDS

致谢

我们感谢本协议所适用的文章的所有作者(Loubiere等人,2016年)。我们感谢FrédéricBienvenu(法国蒙彼利埃功能基因组研究所)的技术支持。我们感谢Filippo Ciabrelli的讨论和帮助,撰写本手稿。

参考

  1. Loubiere,V.,Delest,A.,Thomas,A.,Bonev,B.,Schuettengruber,B.,Sati,S.,Martinez,AM and Cavalli,G。(2016)。&lt; a class = -insertfile“href =”http://www.ncbi.nlm.nih.gov/pubmed/27643538“target =”_ blank“> PRC1蛋白质的协调重新调配可抑制果蝇发育过程中的肿瘤形成。 / a> Nat Genet 48(11):1436-1442。
  2. Spratford,CM和Kumar,JP(2014)。&nbsp; 解剖和 (91):51792。
  3. Strutt,H.和Paro,R。(1997)。黑腹果蝇的多梳组蛋白复合物在不同的靶基因上具有不同的组成。 Mol Cell Biol 17(12):6773-6783
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Loubiere, V., Delest, A., Schuettengruber, B., Martinez, A. and Cavalli, G. (2017). Chromatin Immunoprecipitation Experiments from Whole Drosophila Embryos or Larval Imaginal Discs. Bio-protocol 7(11): e2327. DOI: 10.21769/BioProtoc.2327.
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