Chromatin Extraction from Frozen Chimeric Liver Tissue for Chromatin Immunoprecipitation Analysis

Tissue sampling from human liver chimeric mice²¹ was performed in accordance with the European Union directive 86/609/EEC and approved by the ethical committee of the city and state of Hamburg in accordance with the principles of the Declaration of Helsinki.

1. Reagents preparation

1. Prepare 1.25 M glycine solution in deionized water. Sterile filter with 0.22 µm pore-sized filter. Store at 4 °C.
2. Prepare 5 M sodium chloride (NaCl) solution. Store at room temperature.
3. Prepare CaCl₂ solution: 300 mM CaCl₂ and 10 mM Tris-HCl pH 8 in deionized water. Sterile filter with 0.22 µm pore-sized filter and store at RT.
4. Prepare a 10% Triton X-100 dilution in deionized water. Store at RT.
5. Prepare Tris-EDTA buffer: 1 mM EDTA and 10 mM Tris pH 8 in deionized water. Store at 4 °C.
6. Prepare the following buffers according to the amount required:
   1. Prepare buffer A: 50 mM Hepes-KOH pH 7.5, 140 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 10% Glycerol, 0.5% NP-40 and 0.25% Triton X-100 in deionized water. Sterile filter with 0.22 µm pore-sized filter. Store at 4 °C.
   2. Prepare buffer B: 10 mM Tris-HCl pH 8, 200 mM NaCl, 1mM EDTA, 0.5 mM egtazic acid (EGTA). Sterile filter with 0.22 µm pore-sized filter. Store at 4 °C.
   3. Prepare buffer C: 1% SDS, 10 mM EDTA and 50 mM Tris-HCl pH 8 in deionized water. Sterile filter with 0.22 µm pore-sized filter. Store at RT.
   4. Prepare chromatin dilution buffer: 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.6 mM Tris-HCl pH 8 and 166 mM NaCl in deionized water. Sterile filter with 0.22 µm pore-sized filter. Store at 4 °C.

2. Material preparation

1. Collect dry ice, ice, and liquid nitrogen.
   CAUTION: Handle dry ice and liquid nitrogen with the necessary care to avoid burns.
2. Pre-cool the centrifuge at 4 °C.
   NOTE: This step is important to avoid protein degradation and de-crosslinking during the washing steps as this will reduce the quality of the chromatin.
3. Put a sterile plate on dry ice and let it chill.
   NOTE: Make sure that the plate is big enough to facilitate the cutting process. A 100 mm Petri plate/cell culture dish is recommended.
4. Take out the necessary aliquot of glycine 1.25 M and let it reach RT.
5. Take out the necessary aliquots of buffer A, B and PBS. Add protease and/or deacetylase and phosphatase inhibitors to reach 1-fold concentration and leave them on ice.
6. Take out the necessary aliquot of buffer C leaving it at RT. Do not add the protease and/or deacetylase and phosphatase inhibitors until specified.
7. Take out the necessary aliquot of RT PBS.
   CAUTION: Buffer C contains sodium dodecyl sulfate (SDS). Adopt the appropriate safety measures when preparing the buffer.
   NOTE: SDS precipitates on ice, and protease and deacetylase inhibitors are not stable at RT.
8. Pre-cool the mortar pouring liquid nitrogen in its chamber, strictly following supplier instructions. Cool down the metal pestle in dry ice for at least 5 min.
   NOTE: It is possible to use an alternative mortar to the one proposed. However, the device used in this protocol, due to its peculiar construction, allows to work with small amount of tissue without substantial loss during the pulverization process.
9. Pre-chill the Dounce homogenizer with the associated pestle A on ice.
   ​NOTE: Pestle A has a loose fit with the homogenizer. This allows to obtain a single cell suspension without significant cell lysis.

3. Tissue crosslinking

1. Cut about 50 mg of frozen tissue directly on the dish on dry ice with the help of a scalpel and tweezers.
   NOTE: It is suggested to keep the scalpel at RT, as this will ease the cutting process. Avoid applying too much pressure on the scalpel, as this will increase the risk to scatter tissue pieces outside of the cutting area. To note, 50 mg of tissue (liver in this case) should yield around 5 million cells. Note that the warm blade will thaw the cutting edge. Considering the relatively large size of the tissue piece, however, this should have a limited effect. When smaller pieces are cut, it could be beneficial to use a cold scalpel paying attention to avoid scattering of the tissue.
2. Put the cut tissue in a 1.5 mL tube already chilled on dry ice. Avoid tissue thawing.
3. Move the tube containing the tissue to the mortar, letting it sit there for 5 min.
   NOTE: Letting the sample rest in the mortar decreases its temperature (from -80 °C to -196 °C). This increases its toughness and makes the pulverization step easier.
4. Apply pressure to the sample with the help of the pre-chilled pestle until no more solid crumbles are visible.
   NOTE: It is important to avoid pestle heating by excessive rotational forces, as this will thaw the sample. After every sample pulverization clean the pestle with 70% ethanol (EtOH) and let it chill again on dry ice.
5. Remove the tube containing the sample from the mortar and add 950 µL of ice-cold PBS with the required inhibitors. Pipette gently up and down until the sample is completely resuspended. Proceed immediately to step 3.6.
6. Transfer the tissue suspension to the homogenizer and apply 20-30 strokes with pestle A to obtain a finer suspension. Avoid foaming.
   NOTE: The strokes amount should be optimized according to the tissue consistence. This step dissociates further the small clusters of cells obtained after pulverization. An improper homogenization can affect shearing efficiency.
7. Transfer the homogenate to a new 1.5 mL tube, already prechilled on ice.
8. Centrifuge for 5 min at 1,300 x g at 4 °C and carefully remove the supernatant.
9. Resuspend the pellet completely in 950 µL of RT PBS by gentle pipetting and add 63.6 µL of 16% MeOH-free FA to have a 1% final concentration. Proceed immediately to step 3.10.
   CAUTION: FA is a toxic chemical. Handle it under a fume hood with proper safety measures.
   NOTE: Incomplete resuspension can provoke cell aggregation during the fixation step. This hinders the lysis and the shearing process.
10. Rotate 10 min at RT. Proceed then immediately to step 3.11
    NOTE: Rotation is necessary to avoid aggregates. The amount of time required for fixation should be optimized, according to the target of interest and the sample type. It is important to note that excessive fixation times could hinder proper shearing.
11. Add 113 µL of 1.25 M glycine at RT to obtain a 125 mM final concentration and rotate for 5 min.
    NOTE: Glycine quenches the fixative reaction avoiding over-crosslinking.
12. Centrifuge at 1,300 x g for 3 min at 4 °C.
13. Discard the supernatant and resuspend the pellet carefully by pipetting in 950 µL of ice-cold PBS with the required inhibitors.
14. Centrifuge at 1,300 x g for 3 min at 4 °C.
15. Repeat steps 3.13-3.14 and proceed immediately to the chromatin isolation steps.

4. Chromatin isolation

1. Add 950 µL of Buffer A with the required inhibitors to the pellet. Mix gently by pipetting until the pellet is completely resuspended and rotate 10 min at 4 °C.
   NOTE: This step lyses the fixed single cell suspension, without nuclei lysis. This allows for ridding the sample of cytosolic proteins and RNAs. Prolonging lysis time could be beneficial for tough-to-lyse cells, increasing, though, the handling time of the tissue. At this point it is possible to check the preparation under the microscope after trypan blue/DAPI staining to check the size of the clusters and the presence of single cells. However, the single nuclei may not be easy to appreciate because of the fixed tissue material.
2. Centrifuge at 2,000 x g for 5 min at 4 °C and carefully remove the supernatant.
3. Add 950 µL of Buffer B with the required inhibitors to the pellet. Mix gently by pipetting until the pellet is completely resuspended and rotate 10 min at 4 °C.
   NOTE: This step washes out the lysis buffer from the nuclei preparation to avoid further undesired lysis.
4. Centrifuge at 2000 x g for 5 min at 4 °C. Meanwhile, add the required inhibitors (same as step 2.5) to buffer C.
5. Carefully remove the supernatant.
6. Add 300 µL of RT buffer C to the pellet and pipette vigorously.
7. Vortex the sample for 15-30 s and spin the tube briefly to collect the drops on the lid.
   ​NOTE: This step is important to liberate and lyse the fixed nuclei. To preserve the sample integrity and at the same time avoid SDS precipitation keep the sample prior to sonication in a plastic rack kept on ice to maintain a temperature of 9-11 °C.

5. Chromatin fragmentation

1. Transfer the sample to three clean 0.65 mL sonication-certified tubes ensuring 100 µL of lysed nuclei suspension per tube.
   NOTE: It is possible to use 1.5 mL sonication-certified tubes with a maximum volume of 300 µL. A specific holder for those tubes is needed. 0.65 mL should offer more homogeneous shearing due to the smaller volume of the sample per tube.
2. Sonicate the chromatin for 28 cycles at high intensity with the 30 s ON and 30 s OFF setting. Ensure that the sonicator bath is properly cooled (ice or cooling device).
   NOTE: This step needs optimization in almost every case. The user should keep in mind that increasing shearing time will provide smaller and more homogeneous fragments; however, this may increase the chance to lower chromatin quality. Choose the lowest number of cycles that provides the required fragment size. During the optimization of this step, it is useful to perform nuclear staining to check whether the number of cycles was sufficient to lyse the majority of nuclei.
3. Transfer the sonicated chromatin to a new 1.5 mL tube previously chilled on ice.
4. Add 30 µL of Triton X-100 10% solution and vortex for 5-10 s.
   NOTE: Triton X-100 binds the SDS preventing further precipitation at 4 °C. The final amount of Triton X-100 should always be 1%.
5. Centrifuge at 16,000 x g for 15 min at 4 °C.
6. Transfer the supernatant to a clean 1.5 mL tube pre-chilled on ice.
7. NOTE: The supernatant contains the sheared chromatin and should appear clear. The pellet contains "not shearable" rests and it should remain quite small (mostly brown in case of liver tissue). Look for indication of unsuccessful shearing: chromatin solution that did not become clearer and similar pellet dimensions to the ones from step 4.5.

6. DNA purification

1. Transfer 10-25 µL of sheared chromatin to a new tube and add Buffer C to reach a final volume of 200 µL. Store the rest of the chromatin at -80 °C until further use. If needed, the procedure can be interrupted at this step and the sample stored at -20 °C.
2. Add 8 µL of 5 M NaCl and incubate at least 6 h at 65 °C in a heating block under shaking at 1000 rpm.
   NOTE: This step de-crosslinks the chromatin. It is safer to extend the de-crosslinking overnight when possible. The presence of NaCl makes the process more efficient.
3. Let the samples cool at RT for 5 min and add 2 µL of RNase A.
4. Incubate for 1 h at 37 °C under shaking at 1000 rpm.
5. Remove the samples from the heating block and add 7 µL of 300 mM CaCl₂ and 2 µL of Proteinase K.
6. Set the heating block at 56 °C and incubate for 30 min under shaking at 1000 rpm. Meanwhile prepare one phase-separation tube for every sample by centrifuging them down at 16,000 x g for 1 min at 4 °C.
   NOTE: These special tubes make the phase separation during nucleic acid phenol-chloroform extraction easier.
7. Remove the tubes from the heating block and let them equilibrate at RT for 3 min.
8. Transfer 400 µL of the sample to a previously centrifuged phase-separation tube.
9. Add 400 µL of phenol-chloroform-isoamyl alcohol solution (PCI) and vortex for 5 s.
   CAUTION: PCI is a highly volatile and toxic compound. Please handle it with the necessary safety measures under a fume hood.
10. Centrifuge at 16,000 x g for 5 min at 4 °C.
11. Add 400 µL of Chloroform and vortex for 5 s.
    CAUTION: Chloroform is a highly volatile and toxic compound. Please handle it with the necessary safety measures under a fume hood.
    NOTE: This step washes out possible residues of phenol, that could interfere with downstream PCR applications.
12. Centrifuge at 16,000 x g for 5 min at 4 °C.
13. Transfer 400 µL of the upper phase to a new 1.5 mL tube where 24 µL of 5 M NaCl and 0.75 µL glycogen were added. Briefly vortex.
14. Add 1,055 µL of 100% EtOH and vortex thoroughly. Ensure proper mixing.
15. Incubate at -80 °C for 1 h or at -20 °C overnight (ON).
    NOTE: This step precipitates the sheared DNA; to maximize the yield it is suggested to choose the ON incubation.
16. Centrifuge at 16,000 x g for 30 min at 4 °C.
17. Carefully remove the supernatant paying attention to not dislocate the pellet.
18. Add 500 µL of cold 70% EtOH. Tilt the tube gently to ensure the pellet is washed.
    NOTE: This step is essential to remove salt residues that could have co-precipitated with the nucleic acids. Salts can interfere with other downstream applications.
19. Centrifuge at 16,000 x g for 15 min at 4 °C.
20. Remove carefully the whole supernatant and let the pellet dry at RT.
    NOTE: Incubating the tube on a heating block at 37 °C will reduce the time required for drying.
21. Add 50 µL of Tris-EDTA solution (TE-Buffer) and put the tube on the heating block at 37 °C for 5-10 min under shaking at 300 rpm.
    NOTE: This step ensures the pellet dissolution. The protocol can be paused here, and the sample can be stored at 4 °C for up to 1 week or at -20 °C for longer storage.
22. Perform DNA analysis on 1% agarose gel.

7. DNA size analysis

1. Prepare a 1% agarose gel by mixing 1 g of agarose per 100 mL running buffer (i.e., Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE)). Heat the suspension until the agarose is completely dissolved. Add 10 µL of EtBr for every 100 mL of agarose solution before pouring the gel solution.
   CAUTION: EtBr is a DNA intercalating agent known to be carcinogenic. Please handle it with the necessary safety measures under a fume hood.
   NOTE: EtBr staining (directly in gel or after the run) is strongly suggested. Other DNA-intercalating dyes did not perform well in our hands when working with DNA smears. Narrow loading wells provide a better resolution when compared to wider ones.
2. Mix 10 µL of the sample with 2 µL of 6x Loading Dye. Next, load 10 µL of the sample in the gel and run it until the last band of the loading dye ran for 2/3 of the gel. Make sure to add a DNA ladder.
3. Image the gel and verify if the smear size falls in the range for the desired application.
   If the chromatin passes the quality control, it can be used for downstream applications.