Investigating Physical Chromatin Associations Across the Xenopus Genome by Chromatin Immunoprecipitation

Cross-Linking Proteins to Their Endogenous DNA Binding Sites

• 1. Prepare the embryos.

  • i. Consult Table 1 to estimate the number of embryos required to perform your ChIP experiment.

    • Currently, ∼5–10 million cells expressing the DNA-associated protein of interest are required to create a ChIP-Seq library. For example, at late gastrula stage (Stage 12), ∼200 embryos with ∼40,000 cells each may be enough if the protein is ubiquitously expressed. More embryos are needed accordingly for locally expressed proteins. ChIP-qPCR experiments require only a fraction of these quantities (e.g., ∼100,000–500,000 cells expressing the protein of interest). At gastrula stage, 50 to 100 embryos are generally enough for most ChIP-qPCR experiments assuming that some proteins of interest are only expressed in small parts (up to 1/40th) of the embryo. These empirical numbers may change with varying antibody quality, cross-linking efficiency, and epitope accessibility. See Discussion.

  • ii. Harvest dejellied Xenopus embryos, or parts of them, at the developmental stage of interest.

    • Embryos can be harvested and dejellied according to Sive et al. (2007).

  • iii. Transfer the embryos to a scintillation glass vial and wash them once in the appropriate amphibian medium.

    • We use 0.01× MMR for Xenopus tropicalis embryos or 0.1× NAM for Xenopus laevis.

• 2. Fix whole embryos by adding formaldehyde to a final concentration of 1% in the appropriate amphibian medium (e.g., 260 µL formaldehyde solution [36.5%–38%] in 9 mL amphibian medium). Incubate the embryos in the fixative at room temperature for 20–25 min (X. tropicalis) or 30–35 min (X. laevis).

  • The appropriate fixation time to immobilize proteins on DNA must be determined empirically and varies according to frog species, developmental stage, DNA binding properties, and antibody epitope. The indicated time range is a good starting point for fixing gastrula-stage embryos. Embryos of later stages or dissected parts are normally fixed for a shorter time, such as 15 min (X. tropicalis) or 20–25 min (X. laevis) for late neurula/early tailbud embryos. Xenopus laevis embryos are fixed longer because of their bigger size. See Discussion.

• 3. Remove the fixative by quickly washing embryonic tissue three times with the appropriate ice-cold amphibian medium.

  • This inconspicuous washing step is key for extracting nuclei from cross-linked tissue and then efficiently shearing chromatin (see Discussion). At this stage, embryos are very fragile and should not come into contact with the liquid surface.

  • These fixed embryos can be used immediately (see Step 10); alternatively, they can be frozen and stored for later use as described in Step 4.

• 4. (Optional) Freeze and store the embryos as follows.

  • i. Carefully transfer the embryonic tissue (e.g., groups of 250 embryos or fewer) into prechilled 2-mL microcentrifuge tubes. Remove as much remaining medium as possible from the embryos.

  • ii. Add approximately one volume (equivalent to the volume of embryonic tissue) of ice-cold HEG solution.

  • iii. Allow the embryos to settle to the bottom of the tube and remove the excess HEG.

  • iv. Snap-freeze the embryos in liquid nitrogen and store at −80°C for future use.

Coupling ChIP-Grade Antibody to Magnetic Beads

• At least 1 h before immunoprecipitation (Step 31), magnetic beads of the appropriate type (usually anti-IgG or protein A/G) are blocked and coupled to a ChIP-grade antibody. Use the following guidelines as a starting point for X. tropicalis embryos. For ChIP-qPCR experiments, nonspecific Ig of the antibody isotype are also coupled to the beads to be used as a control.

• 5. Transfer the required volume of resuspended magnetic beads (see Table 2) into a siliconized low-retention 1.5-mL microcentrifuge tube on ice.

• 6. Remove the Dynabeads buffer from the magnetic beads by leaving the tube in its magnetic rack for 20-30 sec and then decanting or withdrawing the buffer with a pipette, being careful not to disturb the beads.

• 7. Wash the beads three times (5 min each) in Dynabeads blocking buffer by rotating the tubes on a rotator (10 rpm) at room temperature. After each wash, remove the buffer as described in Step 6.

• 8. Add the appropriate amount of antibody (or Ig control) to the beads (see Table 2) and adjust the total volume to 400 µL (to ensure mixing) with blocking buffer. Saturate the beads with antibodies by rotating the tubes on a rotator (10 rpm) for 1 h at room temperature or overnight at 4°C.

  • The antibody-saturated beads will keep from several days to weeks at 4°C depending on the antibody, but it is best to use them in Step 28 immediately or within 1–2 d.

Extracting Nuclei from Fixed Embryos

• The extraction of nuclei from fixed embryonic tissue works best with fixation times as outlined in Step 2 and no more than 50–80 X. tropicalis gastrula-stage embryos or 25–40 X. laevis gastrula-stage embryos per milliliter of extraction buffer (E1, E2, and E3). If more embryos are required for a ChIP experiment, it is best to use multiple 2-mL microcentrifuge tubes or 15- or 50-mL conical tubes. Because the yolk content decreases during development, it may be possible to increase the number of embryos per milliliter of extraction buffer after the gastrula stage. During Steps 9–15, keep all biological samples and solutions on ice.

• 9. Supplement the extraction buffers E1, E2, and E3 (Lee et al. 2006) as follows immediately before use.

  • i. Supplement all three buffers with 0.2 mm PMSF and protease inhibitor tablets (1 mini tablet per 10 mL or 1 ultra tablet per 50 mL).

  • ii. Supplement buffer E1 with 1 mm DTT.

  • iii. If it is necessary to block phosphatase activity, to each of the buffers add NaF to a final concentration of 5 mm and Na₃VO₄ to 2 mm.

• 10. Homogenize the fixed embryonic tissue with E1 by pipetting up and down. Centrifuge the homogenate at 1000g for 2–5 min in a refrigerated (4°C) centrifuge, and then aspirate the supernatant and any lipids attached to the wall.

• 11. Resuspend the pellet in E1, keep the suspension on ice for 5–10 min, and then centrifuge and aspirate as in Step 10.

• 12. Resuspend the pellet in E2, and then centrifuge and aspirate as in Step 10.

• 13. Repeat Step 12, but keep the suspension on ice for 5–10 min before centrifugation.

• 14. Resuspend pellet in E3, and then centrifuge and aspirate as in Step 10.

  • The anionic detergents deoxycholate and N-lauroylsarcosine in E3 facilitate solubilization of yolk platelets. This step thus facilitates the separation of yolk from a fairly pure population of cross-linked nuclei (Gentsch et al. 2013). On very efficient extraction of cross-linked nuclei, the nuclear pellet is hardly visible. However, it may appear brown because of some unsolubilized pigment particles. Note that without formaldehyde-mediated cross-linking, these anionic detergents would solubilize the chromatin and this purification step would not work.

• 15. Resuspend the pellet of cross-linked nuclei in a total volume of at least 500 µL of E3 for sonication. If the nuclear suspension appears very viscous, dilute it with E3 to a total volume of 1–3 mL. If several samples were created, pool them at this stage (i.e., before sonication).

Shearing Cross-Linked Chromatin

• Shearing of cross-linked chromatin can be achieved by sonication with a commercially available sonicator. Here we describe the parameters used with a Misonix Sonicator 3000 Ultrasonic Liquid Processor equipped with a tapered 1/16 inch (1.6 mm) microtip. Other laboratories have successfully used different sonicators such as those from Branson or Covaris. When using these sonicators, follow the manufacturers' instructions and optimize the parameters empirically.

• 16. Score and clip a 15 mL polystyrene tube at the 7 mL mark, for use as a vessel for shearing chromatin.

• 17. Transfer the solution with cross-linked nuclei into this conical tube, and keep it chilled during sonication by attaching the tube to a plastic beaker filled with ice water via a short thermometer clamp. With a laboratory jack, move the beaker/tube vertically and horizontally so that the sonicator microtip is immersed in the sample to about two-thirds of the volume depth and properly centered without contacting the tube wall.

  • Off-center positioning of the microtip relative to the tube can cause frothing of the sample.

• 18. Sonicate the sample of cross-linked nuclei. Expose the sample to 10–14 cycles of 30-sec shock waves followed by 1-min pauses to avoid overheating the sample. Start sonication with a power setting of 1.0 W, and change the power setting (depending on volume and ionic strength of sample) to reach 6–12 W for efficient shearing. If the sample begins to froth, pause immediately, reposition the tube, and wait until the froth disappears before resuming sonication.

  • These settings produce an asymmetric DNA fragment distribution in the range of 100–500 bp, peaking at ∼200–300 bp (Fig. 1).

• 19. After sonication, transfer the chromatin solution into microcentrifuge tubes, and remove the debris by centrifuging at full speed (>15,000g) for 10 min at 4°C.

• 20. Transfer the clear supernatant containing solubilized and sheared chromatin to a clean, prechilled 1.5-mL microcentrifuge tube.

• 21. Collect a chromatin sample (∼50 µL) corresponding to ∼400,000 or more nuclei (∼10 gastrula embryos) to check the efficiency of chromatin shearing (see Steps 22–27). Use the remaining chromatin for immunoprecipitation (see Steps 28–38). Keep it at 4°C if it will be used on the same or following day; alternatively, snap-freeze the sample in liquid nitrogen and store it at −80°C for later use.

  • Storage at 4°C or at −80°C does not affect the outcome.

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Figure 1.

Sonication-mediated shearing of cross-linked chromatin from gastrula embryos. The image shows the results of gel electrophoresis of genomic DNA purified from cross-linked chromatin. The chromatin sample was sheared by 10 cycles of sonication (30-sec shock waves followed by 1-min pauses) at 6–9 W.

Checking the Efficiency of Chromatin Shearing

• Efficient shearing of chromatin is important, because the resultant fragment size distribution determines the positional resolution of binding events. This is particularly true for ChIP-qPCR, which does not include a fragment size-selection step before PCR amplification.

• 22. Add at least one volume of fresh SDS elution buffer to the chromatin sample (e.g., 50 µL of SDS elution buffer to 50 µL of solubilized chromatin).

• 23. Add 1/20 volume of 5 m NaCl to the chromatin solution (e.g., ∼5 µL of 5 m NaCl to ∼100 µL of sample) to enhance the reversal of cross-links at 65°C.

• 24. Incubate the chromatin solution for 6–15 h (overnight) in an oven set to 65°C.

• 25. Add proteinase K to the chromatin solution to a final concentration of 200 µg/mL (e.g., ∼1 µL of 20 µg/µL proteinase K to ∼100 µL of sample). Incubate for 2 h at 55°C.

• 26. Purify the DNA fragments using the QIAquick PCR Purification Kit according to the manufacturer's instructions. Elute the DNA with 30 µL of Buffer EB (provided with the kit).

• 27. Add ∼4 µg of RNase A to remove any residual RNA. Incubate for 5 min at 37°C before running the entire sample on a 1.4% agarose gel by electrophoresis.

  • An asymmetric DNA fragment distribution should be visible, mainly within the range of 100–500 bp, peaking at ∼200–300 bp (Fig. 1). If shearing of cross-linked chromatin was successful, continue with Step 28; otherwise, see Troubleshooting.

Immunoprecipitation of Sheared Chromatin

• Use siliconized low-retention 1.5 mL microcentrifuge tubes to collect chromatin at any given step. Use 1.0–1.4 mL of indicated buffer to wash beads at any washing step.

• 28. Wash the beads coupled to antibody (or Ig control) from Step 8.

  • i. Wash the beads twice (5 min each) in Dynabead blocking buffer by rotating the tubes on a rotator (10 rpm) at 4°C. After each wash, remove the Dynabeads buffer from the magnetic beads by leaving the tubes in their magnetic rack for 20–30 sec and then decanting or withdrawing the buffer with a pipette, being careful not to disturb the beads.

  • ii. Wash the beads once (for 5 min) in buffer E2 containing 1% Triton X-100 by rotating the tubes on a rotator (10 rpm) at 4°C. Remove the buffer as in Step 28.i.

• 29. From the sheared chromatin preparation (Step 21), collect a sample that is equivalent to 3%–10% of the ChIP input (e.g., if the 275-µL sample in Step 31 will contain ∼250 µL of sheared chromatin, take ∼25 µL here). Transfer the sample to a new tube and store at 4°C.

  • This sample will be processed further once the ChIP samples are ready for the reversal of cross-links (Step 39), and used as input to create locus-specific qPCR standard curves or ChIP-Seq control libraries.

• 30. To the sheared chromatin from Step 21, add ice-cold 10% Triton X-100 to a final concentration of 1% (e.g., add 55 µL of 10% Triton X-100 to a 500-µL volume of sheared chromatin).

• 31. Add the sheared chromatin from Step 30 to the beads from Step 28. If using an Ig control (for a ChIP-qPCR experiment), add half of the sheared chromatin from Step 30 to the beads coupled to antibody and the other half to the beads coupled to the Ig control (i.e., add 275 µL of sample from Step 30 to each tube). Incubate on a rotator (10 rpm) overnight at 4°C.

• 32. The following day, wash the beads extensively.

  • i. Remove the unbound chromatin from the magnetic beads by leaving the tubes in their magnetic rack for 20–30 sec and then decanting or withdrawing the solution with a pipette, being careful not to disturb the beads.

  • ii. Wash the beads four times (for ChIP-qPCR) or ten times (for ChIP-Seq) (5 min each) with prechilled RIPA buffer by rotating the tubes on a rotator (10 rpm) at 4°C. Remove the buffer as in Step 32.i.

  • iii. Wash the beads once (for 5 min) in prechilled TEN buffer by rotating the tubes on a rotator (10 rpm) at 4°C. Remove the buffer as in Step 32.i.

    • Different washing conditions such as higher salt concentrations (giving higher stringency) can be used to improve the signal-to-noise ratio. The following washing buffers have also frequently been used in the indicated order: low-salt solution buffer, high-salt solution buffer, and LiCl solution buffer.

    • For ChIP-Seq, continue with Step 33; for ChIP-qPCR, skip to Step 36. Steps 33 to 35 help to reduce the number of background fragments in a ChIP-Seq experiment. These steps are not required when performing ChIP-qPCR.

• 33. Resuspend the beads in 50 µL of prechilled TEN buffer, and transfer the bead suspension to a new tube. If several tubes of the same ChIP experiment are in use, pool the samples at this stage.

• 34. Use the magnetic rack to assemble beads at the bottom of the tube.

• 35. Centrifuge the tube at 1000g for 1 min at 4°C, and remove the supernatant while keeping the bottom of the tube adjacent to the magnetic rack.

  • The combination of Steps 34 and 35 ensures that the beads are collected at the bottom of the tube. Centrifugation alone may not be sufficient; some heads can stick to the tube wall in the absence of any detergent.

• 36. Add fresh SDS elution buffer to beads. Use 100 µL for ChIP-qPCR or 200 µL for ChIP-Seq.

  • The recovery of immunoprecipitated DNA may be improved by eluting twice with SDS elution buffer, using either 50 µL (for ChIP-qPCR) or 100 µL (for ChIP-Seq) each time.

• 37. Shake the beads for 15 min in a thermomixer set to 1000 rpm and 65°C.

  • Alternatively, shake the beads every 5 min on a vortex. During the interim, incubate at 65°C.

• 38. Centrifuge the tube at full speed (>15,000g) for 30 sec at room temperature. Transfer the supernatant to a new tube.

  • If the DNA is to be eluted twice, repeat Steps 36–38, and then transfer the supernatant from the second elution to the tube containing the supernatant from the first elution.

Recovery and Extraction of the DNA

• 39. Add at least one volume of fresh SDS elution buffer to the input sample collected at Step 29, typically to a final volume of 100 µL (for ChIP-qPCR) or 200 µL (for ChIP-Seq).

• 40. Add 1/20 volume of 5 m NaCl to all ChIP and input samples (e.g., add ∼5 µL of 5 m NaCl to ∼100 µL of sample).

• 41. Incubate the chromatin solution(s) for 6–15 h (overnight) in an oven set to 65°C.

• 42. Dilute the samples with one volume of TE buffer (e.g., add ∼100 µL of TE buffer to ∼100 µL of sample from Step 41).

• 43. Add RNase A to a final concentration of 200 µg/mL (e.g., add ∼2 µL of 20 µg/µL RNase A to ∼200 µL of sample from Step 42). Incubate for 1 h at 37°C.

• 44. Add proteinase K to a final concentration of 200 µg/mL (e.g., add ∼2 µL of 20 µg/µL proteinase K to ∼200 µL of sample from Step 43). Incubate for 2–4 h at 55°C.

• 45. Extract the DNA fragments using the QIAquick PCR Purification Kit or phenol:chloroform:isoamyl alcohol purification followed by ethanol precipitation.

• To extract DNA using the kit:

  • i. Follow the manufacturer's instructions for the QIAquick PCR Purification Kit.

  • ii. Add 3 M sodium acetate (pH 5.2) as outlined in the manufacturer's instructions (Step 2).

  • iii. Elute the DNA with 30–80 µL of Buffer EB prewarmed to 50°C.

• To extract the DNA using phenol:chloroform:isoamyl alcohol purification and ethanol precipitation:

  • i. Prespin 1.5-mL Phase-Lock Gel Heavy tubes at 12,000g for 30 sec at room temperature.

  • ii. Transfer the DNA samples from Step 44 to the prespun tubes, add one volume of phenol:chloroform:isoamyl alcohol (25:24:1; pH 7.9), and mix the samples by inverting the tubes four to six times.

  • iii. Centrifuge the tubes at full speed (>15,000g) for 5 min at room temperature.

  • iv. Transfer the upper (aqueous) phase to a fresh tube. Add 1/25 volume of 5 m NaCl, two volumes of 100% ethanol, and 15 µg of GlycoBlue. Mix the sample by inverting the tube four to six times.

  • v. Incubate the samples overnight at −20°C.

  • vi. Centrifuge the samples at full speed (>15,000g) for 1 h at 4°C, and then discard the supernatant without disturbing the blue DNA pellet.

  • vii. Wash the pellet with 500 µL of 80% ethanol.

  • viii. Centrifuge the samples at full speed (>15,000g) for 3 min at 4°C, and then discard the supernatant without disturbing the blue DNA pellet.

  • ix. Air-dry the DNA pellet for 5–10 min at room temperature.

  • x. Add 32 µL of molecular-grade water to the dry DNA pellet, and leave samples on ice for at least 2 h to ensure that the DNA is completely dissolved.

    • We prefer to use the QIAquick PCR Purification Kit for ChIP-qPCR because of its high reproducibility and time efficiency, and phenol:chloroform:isoamyl alcohol purification followed by ethanol precipitation for ChIP-Seq because of its superior DNA purification and recovery yield. After extraction, the DNA is ready for qPCR or for making ChIP-Seq libraries (see Discussion). ChIP-isolated DNA samples can be stored at −20°C.

• 46. If the goal is to create ChIP-Seq libraries, quantify 1 µL of the immunoprecipitated DNA and the diluted input DNA using the Qubit Fluorometer (or an equivalent spectroscopic instrument).

  • A total of 5–10 ng of DNA is currently required for creating a robust single-end ChIP-Seq library using the Illumina protocol (see Discussion).