ATAC-Seq Protocol

Tissue Freezing

Snap Freezing

1. Collect the tissue in a sterile cryovial.
2. Immerse the cryovial containing the tissue in liquid nitrogen.
3. Store the vial in a liquid nitrogen storage tank or in a -80℃ freezer for long-term storage.

OCT Embedding and Freezing

1. Before freezing the tissue, arrange cryomolds and label them with a marker.
2. Keep the OCT at room temperature.
3. Put several drops of OCT into the plastic cryomold. Place tissue on top maintaining the correct orientation for cutting. Carefully pour OCT on top of the tissue being careful to avoid bubbles until none of the tissue remains exposed. Hold the cryomold keeping it upright using forceps and introduce it inside liquid nitrogen until the block is frozen. Tissue blocks should be stored at -80℃.

Tissue Sectioning

1. For the histological analysis, cut a 5–10 μm thick cryostat section and mount it on a Superfrost Plus Slide Stain the slides with hematoxylin-eosin (H&E) for histologic examination following standard methods. The tissue can be macrodissected using a sterile blade if necessary.
2. For nuclei isolation, cut 1–2 (50 μm) sections and introduce them into a prechilled 1.5 mL tube.
3. Add 1 mL cold PBS (1×) to remove the OCT.
4. Centrifuge at 1500 ×g for 1 min at 4℃ and discard the supernatant.
5. Repeat steps 1 and 2 until OCT is completely eliminated.

Tissue Homogenization and Cell Lysis

1. After OCT removal, resuspend the tissue section or the snapfrozen tissue in 300 μL cold lysis buffer and dounce the tissue until complete homogenization using disposable pestles in 1.5 mL microtubes.
2. Incubate the tubes for 10 min on ice.
3. Add 1 mL of wash buffer.
4. Pass the homogenized tissue through a 40 μm cell strainer. The flow-through should contain the nuclei.
5. Centrifuge the nuclei at 800 ×g for 10 min at 4℃ and remove supernatant.
6. Wash nuclei with 300 μL wash buffer.
7. Repeat steps 5 and 6 to a total of 2 times.
8. Discard the supernatant and resuspend in 300 μL wash buffer.
9. Count and visualize nuclei with the Countess® II FL Automated Cell Counter counterstained with DAPI reagent. Alternatively, nuclei can be counted using a hemocytometer.
10. If the nuclei preparation still has debris and is not clean enough, repeat steps 4–7.
11. At this step, nuclei are ready to proceed to transposition for bulk and sc analysis. Nuclei can be stored at this point resuspended in 90% FBS/10% DMSO at -80℃ or in liquid nitrogen for extended periods.

Bulk ATAC-Seq

1. Pellet 5000–100,000 nuclei at 800 ×g for 5 min at 4℃.
2. Aspirate supernatant without disrupting the pellet.
3. Resuspend the cell pellet in 50 μL of transposition mix by pipetting up and down 6 times and add 2.5 μL of Tn5 transposase (100 nM final).
4. Incubate the reaction at 37℃ for 30 min in a thermomixer with 850 rpm mixing.
5. Purify using a Qiagen MinElute Kit and elute the transposed DNA in 12 μL Elution Buffer.

DNA Amplification

1. Amplify the transposed DNA preparing a 50 μL reaction containing: 12 μL of the transposed DNA, 2 μL of 5 μM Customized Nextera PCR Primer 1, 2 μL of 5 μM Customized Nextera PCR Primer 2, 0.3 μL of 100× SYBR Green I, 25 μL of NEBNext High-Fidelity 2× PCR Master Mix, and 8.7 μL of nuclease-free water.
2. Run the following PCR program: 1 cycle of 5 min at 72℃ and 30 s at 98℃ followed by N cycles of 10 s at 98℃, 30 s at 63℃, and 1 min at 72℃.
3. The number of cycles depends on the starting number of transposed nuclei. The previously described protocol (OMNI) recommends the performance of an initial 5 cycles reaction followed by a qPCR side reaction to monitor the amplification reaction to estimate the number of additional cycles to be added. However, to facilitate the calculation of cycles required to amplify: add 15 cycles when starting from <10,000 nuclei; add 13–14 cycles from 10,000–50,000 nuclei; add 12–13 cycles for 50,000–100,000 nuclei.
4. Proceed to library sequencing, initial analysis of the sequences, and quality control (QC).

Reference:

1. Cejas P, Long H W. High-Resolution ATAC-Seq Analysis of Frozen Clinical Tissues[M]//Chromatin. Humana, New York, NY, 2022: 259-267.