ChIP-Seq Sample Preparation Protocol

Harvesting Material and CrossLinking

1. Animal tissue: Transfer the fresh or flash-frozen tissue (5–30 mg) to a sterile 1,5 mL tube and add a small volume of ice-cold PBS + protease inhibitors (250 μL to 1.5 mL when starting with 5 mg and 30 mg of tissue, respectively) to disrupt the tissue with a tissue homogenizer. Do not let the volume exceed 1.2 mL; if this occurs, split the sample into two tubes.
2. Complete the volume of homogenized tissue with ice-cold PBS + protease inhibitors (1–6 mL scale up proportionally to the initial amount of tissue), and transfer to an ice-cold 15 mL tube.
3. Cells in suspension: Start with 10⁷ cells (maximum of 5 × 10⁷ cells) that are pelleted and resuspend them in 10 mL of ice-cold PBS.
4. Adherent cells: Trypsinize and collect the cells (10⁷ cells, up to 5 × 10⁷ cells) by centrifugation, remove the supernatant, and add 10 mL of ice-cold PBS.
5. For single cross-linking: Add formaldehyde to a final concentration of 1% and immediately incubate the samples under gentle rotation for 10 min, at room temperature.
6. Quench the reaction by adding glycine to a final concentration of 150 mM and incubate the samples under gentle rotation for 5 min, at room temperature.
7. For double cross-linking: Add EGS (ethylene glycol bis(succinimidyl succinate)) to a final concentration of 1.5 mM and place the samples under gentle rotation for 30 min at room temperature.
8. Add formaldehyde to a final concentration of 1% and incubate with gentle rotation for 10 min at room temperature.
9. Quench the reaction by adding glycine to a final concentration of 150 mM and incubate the samples gently rotating for 5 min, at room temperature.
10. Harvest the tissue or cells by centrifugation at 2000×g for 10 min at 4 °C.
11. Remove the supernatant and add an equal volume of ice-cold PBS. Flick the tube until dislodging the cell pellet.
12. Harvest the tissue or cells by centrifugation at 2000×g for 10 min at 4 °C. At this point, samples can be flash-frozen on liquid nitrogen and stored at -80 °C for up to a month. Always thaw tissue samples on ice (it may take a few hours if the pellets are large).

Chromatin Isolation and Sonication

1. Resuspend the pellet in cell lysis buffer. For a small pellet (10⁷ cells or 5 mg of tissues generate a pellet that is smaller than 50 μL—use the markers on the 1.5 mL tube for reference), add 1 mL of cell lysis buffer. For larger pellets, add 5 mL of cell lysis buffer and pipette up and down until the pellet is completely dissolved.
2. Incubate on ice for 10 min.
3. When lysing tissues: transfer the tissue suspension to a 2 mL Dounce homogenizer (kept on ice) and complete the tissue disruption with the pestle B (20 strokes). Transfer the lysate to a fresh 1.5 mL or 15 mL tube (according to the volume of the cell lysis buffer used on step 6). Wash the homogenizer with 50 μL of cell lysis buffer and combine both volumes.
4. Centrifuge the lysate at 2000×g for 5 min at 4 °C.
5. Remove the supernatant and add the nuclear lysis buffer. For small pellets, add 500 μL of buffer, and for larger pellets, add up to 3 mL of nuclear lysis buffer. Incubate on ice for 10 min.
6. Reserve an aliquot (10–15 μL) of the chromatin solution and keep on ice. This will be used as the non-sonicated control which is used to compare with the sonicated chromatin to verify the efficiency of sonication.
7. Sonicate your samples. As sonication is dependent on cell type, tissue composition, and sonicator model, this step requires optimization. When using a Covaris E220 sonicator with 1 mL AFA fiber tubes (up to 1.2 × 10⁷ cells), we recommend the following settings as a starting point: (PIP = 75, Duty factor = 2%, CPB = 200, time = 1 to 5 min).
8. Pipette two aliquots (10–15 μL) of the sonicated samples, one to determine the efficiency of sonication and another for DNA quantification. Keep the samples on ice.
9. Centrifuge samples at 18,000×g for 10 min at 4 °C to remove the debris. Transfer the supernatant to a fresh 1.5 mL tube and keep on ice. At this point, the chromatin can be flash-frozen on liquid nitrogen and stored at -80 °C for up to a month. Always thaw the chromatin on ice as described above.
10. Add 10 μg of RNAse A to all the reserved aliquots and incubate for 30 min at 37 °C.
11. Add 20 μg of proteinase K to all the reserved aliquots and incubate for 1 h at 65 °C.
12. Boil the aliquots for 10 min at 95 °C to reverse the cross-links and allow the samples to cool down to room temperature.
13. To verify the efficiency of DNA sonication, analyze the DNA fragment size on a 1% agarose gel. If samples are being submitted to NGS, fragment size should range from 200 to 500 bp. If the method of analysis is qPCR, fragments should range from 500 to 1000 bp.
14. Quantify the aliquot reserved for DNA quantification, using the QIAquick PCR Purification Kit. The eluted DNA can be quantified on a NanoDrop spectrophotometer.