Chromatin Immunoprecipitation Sequencing (ChIP-seq) Protocol

Preparation of Cell Suspension and Tissue Lysate

Trypsinize the cultured adherent cells and suspend them in PBS. Alternatively, the cells can be scraped off the culture dish after fixation. Typically, 10⁶–10⁷ cells are used for a single IP; however, the optimal cell number varies depending on the antibody used. For preparing tissue lysates, snap freeze freshly isolated tissue sections (30–100 mg) in liquid nitrogen, and then homogenize them in PBS using a Dounce homogenizer (1–2 mL) with a loose pestle.

Chromatin Fixation and Fragmentation

Histone Modifications

1. Add 1/10 volume of fixation buffer to the cell suspension or tissue lysate and incubate at 25℃ for 5–15 min with mild mixing or rotation.
2. Add 1/10 volume of 1.5 M glycine and incubate at 25℃ for 3 min with a mild mixing or rotation.
3. Spin down and wash twice with PBS.
4. Resuspend the cell pellet in lysis buffer and incubate on ice for 15 min. Homogenize the cells ten times with a syringe equipped with a 21–23G needle. Count nuclei using a hemocytometer or by DAPI staining. Spin down the nuclei for 5 min at 4℃.
5. Resuspend the pellet in SD Slysis buffer at a density of 1–2×10⁶ nuclei/100μL, and incubate on ice for 5 min.
6. Transfer the nuclei suspension to a Picoruptor tube (100–300 μL/tube). Sonication condition: 5–30 cycles (30 sec on/30 sec off) at 4℃.
7. Centrifuge the mixture at 13,000 ×g for 5 min, at 4℃, and collect the supernatant for the subsequent IP steps.

Histone Modifiers

1. Dual crosslink: Prior to formaldehyde crosslink, suspend the cells/tissue lysate in 2 mM DSG, and incubate at 25℃ for 5 min.
2. Sonication: Suspend the nuclei in 1×RIPA buffer, which contains 0.1% SDS, instead of SDS lysis buffer. The optimal sonication time may be longer (up to 50 min) than that of the single-crosslink method.

Immuno-precipitation

1. Preclear: Dilute the fragmented chromatin solution ten times with ChIP dilution buffer. Add Dynabeads Protein A or G at 20μL/mL and rotate at 4℃ for 1h. Place the tube on a magnetic rack for 5 min and collect the supernatant. At this point, chromatin from 1–4×10⁶ cells will be solubilized per 1mL.
2. Antibodies: Keep 10% of the input for subsequent DNA purification. Add antibodies specific to histone modification or modifier at 4–10 μg/mL. A parallel sample adding an equivalent amount of control IgG should be prepared. Rotate overnight at 4℃.
3. Beads pull down: Add 20 μL of Dynabeads Protein A or G and rotate at 4℃ for 2–3h.
4. The beads should be washed five times with RIPA buffer (low salt), RIPA buffer (high salt), and LiCl buffer and twice with TE. Between each wash, mix by rotation for 3–5 min, spin down, and place on a magnetic rack.
5. Reverse crosslinking: Suspend the beads in 200 μL of direct elution buffer. To the input samples, add ChIP dilution buffer to make the total volume of 192 μL, and then add 8 μL of 5M NaCl. Incubate at 65℃ for at least 4h.

DNA Purification

1. Add 1 μL of RNase A, and incubate at 37℃ for 30 min.
2. Add 1 μL of Proteinase K, and incubate at 4℃ for 1 h.
3. Add an equal volume of PCI, vortex, and centrifuge at 13,000 ×g for 5 min. Collect the aqueous phase. For the back extract, add TE/NaCl to the original tube, and repeat the extraction (the aqueous phase combined will sum up to approximately 400 μL).
4. Add 1 μL of glycogen and 900 μL of 100% ethanol. Vortex and incubate at -20℃ for 5 min. Centrifuge at 13,000 ×g for 5 min and discard the supernatant. Wash the pellet with 70–80% ethanol.
5. Suspend the pelleted DNA in 50–100 μL of TE.

NGS Library Prep

1. Prior to library preparation, analyze the size distribution of DNA fragments using a TapeStation or a Bioanalyzer instrument. If neither is available, perform agarose gel electrophoresis combined with SYBR Gold staining which enhances the detection of low-concentration DNA.
2. For library reparation, use an appropriate DNA library preparation kit according to the manufacturer's instructions. We use 20–30 ng of IP DNA or 50–100 ng of input DNA for each library. For library amplification, 10–15 PCR cycles are typically used.
3. It is important to confirm that the size distributions are equivalent between the libraries generated. The amount of library DNA should be quantified using a qPCR-based method. Gen-Next NGS Library Quantification Kit may be used.

Reference:

1. Pei J, van den Dungen N A M, Asselbergs F W, et al. Chromatin Immunoprecipitation Sequencing (ChIP-seq) Protocol for Small Amounts of Frozen Biobanked Cardiac Tissue[M]//Chromatin. Humana, New York, NY, 2022: 97-111.