Jan 15, 2026

Public workspaceCUTAC for FFPE Slides

DOI
https://dx.doi.org/10.17504/protocols.io.5jyl88pp7l2w/v1
  • Steven Henikoff1,
  • Yiling Xu1
  • 1Fred Hutchinson Cancer Center
Yiling Xu
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DOI: https://dx.doi.org/10.17504/protocols.io.5jyl88pp7l2w/v1
External link: https://www.biorxiv.org/content/10.64898/2025.12.24.696395v1
Protocol Citation: Steven Henikoff, Yiling Xu 2026. CUTAC for FFPE Slides. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl88pp7l2w/v1
Manuscript citation:
Xu Y, Ahmad K & Henikoff S (2026) Chromatin profiling for everyone: FFPE-CUTAC for the theory and practice of modern molecular biology. Frontiers in Epigenetics and Epigenomics, In press.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 15, 2025
Last Modified: January 15, 2026
Protocol Integer ID: 224804
Keywords: FFPE, CUT&Tag, FFPE-CUTAC, cutac for ffpe slide, ffpe slide, slide only version of the cutac, ffpes method, ffpe, slide, cutac
Funders Acknowledgements:
Howard Hughes Medical Institute
Abstract
This is the on-slide only version of the CUTAC for FFPEs method (https://dx.doi.org/10.17504/protocols.io.14egn292zg5d/v4).

Figure 1: On-slide FFPE-CUTAC. Schematic of the protocol.

Troubleshooting
Reagent Setup for 8 FFPE Slides in 1 Coplin Jar
30m
50% Ethanol for rehydration (50mL)
10% Ethanol for rehydration (50mL)
80% Ethanol for SPRI beads clean-up (20mL)

Cross-link reversal buffer (800 mM Tris-HCl pH8.0, 0.2 mM EDTA) (50 mL)
40mL 1M Tris-HCl pH8.0
10mL dH2O
20uL 0.5M EDTA

Triton-Wash buffer (50 mL)
1 mL 1 M HEPES pH 7.5
1.5 mL 5 M NaCl
250 µl 10% Triton-X100
12.5 μl 2 M spermidine
20 µL 0.5M EDTA
bring the final volume to 50 mL with dH2O
add 1 Roche Complete Protease Inhibitor EDTA-Free tablet
Store the buffer at 4 °C for up to 2 days.
**Optional: To completely prevent bacterial contamination during long incubations and storage of Triton-wash buffer, add Sodium azide to 0.02% (100 µl 10% -> 50 mL), but handle this toxic chemical carefully, and wear a mask when weighing it out. (10% Sodium Azide: 1g Sodium Azide in 10mL dH2O)

Primary antibody solution (1:25) (250 uL)
10 µl primary antibody - RNA Polymerase II-Ser5p (Cell Signaling Technologies (D9N5I) mAb #13523)
240 µl Triton-Wash buffer

Secondary antibody solution (1:25) (250 uL)
10 µl secondary antibody - guinea pig anti-rabbit (Antibodies Online)
240 µl Triton-Wash buffer

Protein A(G)-Tn5 solution (1:25) (250 uL)
12.5 µl Protein A(G)-Tn5 (Epicypher cat. no. 15-1117)
237.5 µL Triton-Wash buffer

CUTAC-DMF Tagmentation buffer (10 mM TAPS, 5 mM MgCl2, 20% DMF) (50 mL)
39.3 mL dH2O
10 mL N,N-dimethylformamide
0.5 mL 1 M TAPS pH 8.5
250 µl 1 M MgCl2
Store the buffer at 4°C for up to 1 week.

TAPS wash buffer (10 mM TAPS, 0.2 mM EDTA) (50mL)
0.5 mL 1 M TAPS pH 8.5
49.5 mL dH2O
20 µl 0.5 M EDTA

1% SDS / Thermolabile Proteinase K Release solution (100uL)
10 µl 10% SDS
1 µl 1 M TAPS pH 8.5
79 µl dH2O
Just before use add 10 µL Thermolabile Proteinase K (NEB cat. no. P8111S).

5% Triton Mix (1mL)
500 µl 10% Triton-X100
500 µl dH2O
Store at room temperature

Deparaffinization and Cross-link Reversal
1h 35m
Deparaffinization: place slides in a Coplin jar containing SafeClear II Xylene Substitute (Fisher scientific cat. no. 11-002-233) and hold for 10 min at room temperature.
10m
Rehydrate tissue with ethanol washes.
Transfer slides to a Coplin jar containing 100% ethanol and hold for 3-5 min.
5m
Transfer slides to a Coplin jar containing 50% ethanol and hold for 3-5 min.
5m
Transfer slides to a Coplin jar containing 10% ethanol and hold for 3-5 min.
5m
Cross-link reversal: transfer slides to a plastic Coplin jar with lid containing cross-link reversal buffer and incubate at 85C for 1 hr.

Note
Incubations as long as overnight at 80-90C in cross-link reversal buffer have yielded high-quality results similar to results using 1 hr incubations.

Note
Use ~5 µm FFPE sections, which are optimal for this protocol. Ensure sections are mounted on charged glass slides to prevent tissue loss during incubation steps.

1h
Overnight
Take out the plastic Coplin jar and put it into iced cold water to cool down to ~room temperature.

10m
On-slide FFPE-CUTAC Incubation with Primary Antibody
1h 50m
Remove each slide from the slide holder. Drain excess liquid by placing it on a small stack of paper towels. Wick off as much residual liquid as possible from both sides of the slide with a Kimwipe without disturbing the tissue. Place tissue-side up on a dark surface for visibility.
Carefully pipette ~20-40µl primary antibody solution over the tissue. Solution volume should be adjusted based on the size of the tissue.
15m
Cover the clear portion of the slide with a rectangle of plastic wrap (see 7.1 for preparation) using surface tension to spread the liquid, while excluding large bubbles and wrinkles on the tissue.
Prepare a staining dish with some moist paper towel placed at the bottom.
Place covered slides separated in the rack of this staining dish, which can be used as a "moist chamber".

Figure 2: Examples of plastic wrap covered slides for incubations. Plastic wraps should be flat. Bubbles are OK in the area without any tissue. If plastic wrap rectangles are larger than the slides, fold along the edge.

Figure 3: An example of a "moist chamber": a staining chamber with wet paper towel at the bottom.

15m
The method to prepare wrinkle-free plastic wrap rectangles:

  • Place a sheet of clean, letter-size printer paper on a flat surface. Lay a piece of plastic wrap on top, followed by a second sheet of printer paper, creating a paper–plastic–paper “sandwich.”
  • Press carefully to ensure the plastic wrap lies completely flat between the sheets. This “paper sandwich” prevents wrinkles and makes cutting easier. (If more rectangles are needed, add additional paper sandwich layers of plastic wrap and printer paper.)
  • Cut the sandwich to size using scissors, a razor blade, or a paper trimmer. For general use, cut rectangles approximately 2.5 cm × 4 cm, which will fit most tissue areas on slides.
  • Store prepared rectangles flat in a dust-free environment until use.
Figure 4: An example of pre-cut plastic wrap rectangle "sandwiches", stored in a small box with lid.


















Note
Any plastic wrap will seal adequately, but we recommend food service film on a heavy 2000 foot roll (e.g. Reynolds 912) for ease of pulling out wrap with both hands. The ones with slide cutters also help (e.g. Kirkland Strech-tite). Some kitchen wraps (Saran and Glad) are not as smooth and will be more difficult to work with.

20m
Incubate at room temperature for at least 1 hr.

Pause point: Samples may be held overnight at 4 °C in this step.

1h
Pause
Incubation with Secondary Antibody
1h 30m
Remove plastic wrap and gently rinse slide by pipetting 1 mL Triton-Wash buffer dropwise over the top of the slide without disturbing the tissue.
10m
Drain excess liquid by placing slides on a small stack of paper towels. Wick off residual liquid from both sides of the slide with a Kimwipe. Place tissue-side up on a dark surface for visibility.
Carefully pipette ~20-40µl secondary antibody solution over the tissue. Solution volume should be adjusted based on the size of the tissue.
10m
Cover the clear portion of the slide with a rectangle of plastic wrap using surface tension to spread the liquid, while excluding large bubbles and wrinkles on the tissue.
Place covered slides separated in the rack of the moist staining dish used in the previous step.
10m
Incubate at room temperature for at least 1 hr.

Pause point: Samples may be held overnight at 4 °C in this step.

1h
Pause
Binding Protein A(G)-Tn5 Adapter Complex
1h 30m
Remove plastic wrap and gently rinse slide by pipetting 1 mL Triton-Wash buffer dropwise over the top of the slide without disturbing the tissue.
10m
Drain excess liquid by placing slides on a small stack of paper towels. Wick off residual liquid from both sides of the slide with a Kimwipe. Place tissue-side up on a dark surface for visibility.
Carefully pipette ~20-40µl Protein A(G)-Tn5 solution over the tissue. Solution volume should be adjusted based on the size of the tissue.
10m
Cover the clear portion of the slide with a rectangle of plastic wrap using surface tension to spread the liquid, while excluding large bubbles and wrinkles on the tissue.
Place covered slides separated in the rack of the moist staining dish used in the previous step.
10m
Incubate at room temperature for at least 1 hr.

Pause point: Samples may be held overnight at 4 °C in this step.
1h
Pause
Tagmentation
1h 40m
Remove plastic wrap and gently rinse slide by pipetting 1 mL Triton-Wash buffer dropwise over the top of the slide without disturbing the tissue.
10m
Drain on a small stack paper towel, and transfer to a Coplin jar containing TAPS wash buffer for 10 min.
10m
Remove slides and drain on a small stack paper towel.
Place them into a plastic Coplin jar containing cold Tagmentation buffer.
5m
Incubate 1 hr in a water bath at 55°C.
1h
Take out the plastic Coplin jar and put it into iced cold water to cool down to ~room temperature.
5m
Transfer the slides into a Coplin jar containing TAPS wash buffer to hold.

Pause point: Samples may be held overnight in TAPS wash buffer at 4 °C in this step.
10m
Pause
Dissection and Fragment Release
2h
Remove each slide from the slide holder. Drain excess liquid by placing it on a small stack of paper towels. Wick off as much residual liquid as possible from both sides of the slide with a Kimwipe without disturbing the tissue. Place tissue-side up on a dark surface for visibility.

Dissect or directly scrape off the tissue into a PCR tube containing ~5 µL 1% SDS / Thermolabile Proteinase K solution.
  • For most samples, preload each tube with 4 µL of solution, then use an additional 1–2 µL to wet the tissue to facilitate scraping.
  • For very small tissue sections, preload the tube with the full 5 µL and wet the blade with a small amount of solution to scrape.
  • For larger pieces of tissue, subdividing into smaller portions is strongly recommended to reduce input amount and improve downstream PCR success. See below for the two options for subdividing tissues.

Note
Recommended Tools:
Use a single-edge razor blade for large sections, a fine scalpel for very small or delicate sections, and fine-tip forceps (e.g., Dumont #5) to transfer tissue into tubes.

30m
Option 1: Region-Specific Dissection on Slides

Use this approach when collection of defined tissue regions is required (e.g., based on staining, morphology, or area of interest).

  • Dissect the tissue directly on the slide into smaller pieces and transfer each portion into a separate PCR tube preloaded with 1% SDS / Thermolabile Proteinase K solution.

Option 2: Scrape off Tissue, Then Split Before PCR

Use this approach when region-specific collection is not required and bulk material is sufficient.

  • Scrape the entire tissue section from the slide and transfer it into a PCR tube preloaded with 1% SDS / Thermolabile Proteinase K solution.
  • For very large sections, proportionally scale up the lysis buffer (e.g., 10 µL 1% SDS / ProtK solution requires 30 µL 5% Triton X-100 in the quenching step).

Note
Following this approach, for downstream PCR, dilute a desired portion of the supernatant with water. This approach provides better control of input amount and allows the remaining supernatant to be archived for future use if needed.

Incubate at 37ºC for 30 min and 58ºC for 30 min to release pA-Tn5 from the tagmented DNA.
1h 30m
When the samples are still on the thermocycler, add 15 µL 5% Triton-X100 to each reaction to quench the SDS. Continue incubation at 37 °C for 30 min to complete quenching.
PCR
1h 20m
Briefly vortex each tube and perform a quick spin.

  • If Option 1 (Step 23.1) was used, proceed directly to the next step (Step 27).
  • If Option 2 (Step 23.2) was used, dilute a desired volume of supernatant with nuclease-free water to make a total volume of 20 µL (e.g., 1 µL + 19 µL water, 5 µL + 15 µL water).

20m
Add 2 µl of 10 µM Universal or barcoded i5 primer + 2 µl of 10 µM barcoded i7 primers, using a different barcode pair for each sample. Vortex, quick spin, and place tubes in a metal tube holder on ice.

Note
Indexed primers are described by Buenrostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523:486 (2015). We do not recommend Nextera or NEB primers, which might not anneal efficiently using this PCR protocol.

Add 25 µl NEBNext High-Fidelity 2X PCR Master Mix (non-hot-start). Vortex to mix, and perform a quick spin. Place the tubes on the thermocycler and proceed immediately with the PCR.

PCR program - 30s annealing and 1m extension for 13 cycles:
Cycle 1: 58°C for 5 min (gap filling)
Cycle 2: 72°C for 5 min (gap filling)
Cycle 3: 98°C for 5 min
Cycle 4: 98°C for 10 sec
Cycle 5: 63°C for 30 sec
Cycle 6: 72°C for 1 min
Repeat Cycles 4-6 12 times
Hold at 8 °C
1h
Remove each slide from the slide holder. Drain excess liquid by placing it on a small stack of paper towels. Wick off as much residual liquid as possible from both sides of the slide with a Kimwipe without disturbing the tissue. Place tissue-side up on a dark surface for visibility.

Dissect or directly scrape off the tissue into a PCR tube containing ~5 µL 1% SDS / Thermolabile Proteinase K solution.
  • For most samples, preload each tube with 4 µL of solution, then use an additional 1–2 µL to wet the tissue to facilitate scraping.
  • For very small tissue sections, preload the tube with the full 5 µL and wet the blade with a small amount of solution to scrape.
  • For larger pieces of tissue, subdividing into smaller portions is strongly recommended to reduce input amount and improve downstream PCR success. See below for the two options for subdividing tissues.

Note
Recommended Tools:
Use a single-edge razor blade for large sections, a fine scalpel for very small or delicate sections, and fine-tip forceps (e.g., Dumont #5) to transfer tissue into tubes.

Option 1: Region-Specific Dissection on Slides

Use this approach when collection of defined tissue regions is required (e.g., based on staining, morphology, or area of interest).

  • Dissect the tissue directly on the slide into smaller pieces and transfer each portion into a separate PCR tube preloaded with 1% SDS / Thermolabile Proteinase K solution.

Option 2: Scrape off Tissue, Then Split Before PCR

Use this approach when region-specific collection is not required and bulk material is sufficient.

  • Scrape the entire tissue section from the slide and transfer it into a PCR tube preloaded with 1% SDS / Thermolabile Proteinase K solution.
  • For very large sections, proportionally scale up the lysis buffer (e.g., 10 µL 1% SDS / ProtK solution requires 30 µL 5% Triton X-100 in the quenching step).

Note
Following this approach, for downstream PCR, dilute a desired portion of the supernatant with water. This approach provides better control of input amount and allows the remaining supernatant to be archived for future use if needed.

Incubate at 37ºC for 30 min and 58ºC for 30 min to release pA-Tn5 from the tagmented DNA.
When the samples are still on the thermocycler, add 15 µL 5% Triton-X100 to each reaction to quench the SDS. Continue incubation at 37 °C for 30 min to complete quenching.
Post-PCR Clean-up
40m
After the PCR program ends, remove tubes from the thermocycler, vortex to mix, and add 65 µL of SPRI beads (ratio of 1.3 µL of SPRI beads to 1 µL of PCR product). Mix by pipetting up and down or vortexing. Let sit at room temperature 5-10 min.
10m
Place on the magnet stand for a few minutes to allow the solution to clear. Remove and discard the supernatant.
5m
Keeping the tubes in the magnet stand, add 200 µL of fresh 80% ethanol.
Let sit for 30 seconds. Remove and discard the supernatant.
Repeat the 80% ethanol wash step one more time.
5m
Perform a quick spin and remove the remaining supernatant using a 20uL pipette, avoiding air drying the beads by proceeding immediately to the next step.
5m
Remove from the magnet stand, add 22 µl 10 mM Tris-HCl pH 8, vortex and quick spin.
Let sit for at least 5 min to elute the DNA.
10m
Place on the magnet stand for a few minutes to allow the solution to clear. Transfer the supernatant to a clean low-bind 1.5 mL tube with a pipette, avoiding transfer of beads.

5m
Tapestation Analysis and DNA Sequencing
Determine the size distribution and concentration of libraries by capillary electrophoresis using an Agilent 4200 TapeStation with D1000 reagents or equivalent.

Expected result

Figure 5: A TapeStation gel image from an on-slide FFPE CUTAC experiment.

Figure 6: Pictures of the samples in figure 5 for reference. They were all split into smaller portions.


Mix barcoded libraries to achieve equal representation as desired aiming for a final concentration as recommended by the manufacturer. After mixing, perform an SPRI bead cleanup if needed to remove any residual PCR primers.

Note
For a pool of FFPE-CUTAC samples, we usually do a double-sided SPRI cleanup (0.5x then 0.8x), which has worked well to remove both residual primers and higher molecular weight material.

Here’s an example for a 558uL pool vol:
  • Add 0.5x bead vol: 558 x 0.5 = 279 uL
  • Mix & incubate 10 min
  • On magnet 5 min
  • Transfer supernatant to new tube, measuring vol that’s transferred (810 uL in this case)
  • Add 0.8x bead vol: 432 uL beads
(Vpoolx 0.8x (Vsup / (V0.5x + Vpool)) = 558 x 0.8 x (810/837) = 432 uL
  • Mix & incubate 10 min
  • On magnet 5 min
  • Wash 2 times with fresh 80% ETOH
  • Elute in 32 uL 10 mM Tris-HCl pH 8

Note
We use the quantification by Tapestation to estimate library concentration and dilute each library to 4 nM (or the concentration specified for Illumina library submission at the sequencing core that will process your sample) before pooling based on fragment molarity in the 175-500 bp range.

Perform paired-end Illumina sequencing on the barcoded libraries following the manufacturer’s instructions.
Note
We currently use paired-end 50x50 sequencing on an Illumina NovaSeq X+, obtaining ~1250 million total mapped reads.

Protocol references
Xu Y, Ahmad K & Henikoff S (2026) Chromatin profiling for everyone: FFPE-CUTAC for the theory and practice of modern molecular biology. Frontiers in Epigenetics and Epigenomics, In press.

Henikoff, S. et al. RNA polymerase II at histone genes predicts outcome in human cancer. Science 387, 737-743 (2025).

Henikoff, S. et al. Epigenomic analysis of Formalin-fixed paraffin-embedded samples by CUT&Tag. Nat Commun 14, 5930 (2023).