Simplified Epigenome Profiling Using Antibody-tethered Tagmentation

We previously introduced Cleavage Under Targets & Tagmentation (CUT&Tag), an epigenomic profiling method in which antibody tethering of the Tn5 transposase to a chromatin epitope of interest maps specific chromatin features in small samples and single cells. With CUT&Tag, intact cells or nuclei are permeabilized, followed by successive addition of a primary antibody, a secondary antibody, and a chimeric Protein A-Transposase fusion protein that binds to the antibody. Addition of Mg++ activates the transposase and inserts sequencing adapters into adjacent DNA in situ. We have since adapted CUT&Tag to also map chromatin accessibility by simply modifying the transposase activation conditions when using histone H3K4me2, H3K4me3, or Serine-5-phosphorylated RNA Polymerase II antibodies. Using these antibodies, we redirect the tagmentation of accessible DNA sites to produce chromatin accessibility maps with exceptionally high signal-to-noise and resolution. All steps from nuclei to amplified sequencing-ready libraries are performed in single PCR tubes using non-toxic reagents and inexpensive equipment, making our simplified strategy for simultaneous chromatin profiling and accessibility mapping suitable for the lab, home workbench, or classroom.

Despite the utility of chromatin accessibility mapping, the mechanistic basis for chromatin accessibility itself has remained incompletely understood. In contrast to the simplistic designation of chromatin as being "open" or "closed," recent work has shown that the median difference between an accessible and a non-accessible site in DNA is estimated to be only ~20%, with no sites completely accessible or inaccessible in a population of cells (Chereji et  In our original CUTAC study, we described three different modifications of the CUT&Tag-direct protocol for accessible site mapping: tagmentation in MgCl2 with a 20-fold dilution of 300 mM NaCl and pA-Tn5 (or commercial pAG-Tn5 with both Protein A and Protein G IgG specificities), removal of excess pAG-Tn5 before low-salt tagmentation, and low-salt tagmentation following the 300 mM wash step. We have adopted post-wash tagmentation, which follows the same steps as in the original CUT&Tag-direct protocol (Kaya-Okur et al., 2020), changing only the tagmentation buffer composition. As reported here, the application of this CUTAC protocol to the initiation form of RNAPII results in precise chromatin accessibility maps with exceptionally high signal-to-noise. The improvement obtained by tethering to the 4 www.bio-protocol.org/e4043

Figure 2. Scheme for simultaneous CUT&Tag and (H3K4me2 or RNAPIIS5P) CUTAC.
CUT&Tag-direct is performed in situ in single PCR tubes with Concanavalin A (ConA) bead-bound nuclei that remain intact throughout the protocol during successive liquid changes, incubations and washes, 12 cycles of PCR amplification, and one SPRI bead cleanup. CUTAC is performed identically except that low-salt conditions are used for tagmentation. H3K4me2 CUTAC maps accessible sites near H3K4me2/3-marked (starred) nucleosome tails, which are methylated by the conserved Set1 lysine methyltransferase. The complex that includes Set1 associates with the initiation form of RNAPII, which is heavily phosphorylated on Serine-5 of the heptameric C-terminal domain repeat units on the largest RNAPII subunit (RNAPIIS5P). For RNAPIIS5P CUTAC, pA-Tn5 is anchored directly to RNAPIIS5 phosphates (starred). Whereas CUT&Tag is suitable for any chromatin epitope, CUTAC is specific for H3K4me2, H3K4me3, and RNAPIIS5P. The only other difference between the protocols is that tagmentation is performed in the presence of 300 mM NaCl for CUT&Tag and in a low ionic strength buffer for CUTAC.  5. Resuspend in 1/2 volume (relative to starting culture) of ice-cold NE1 buffer with gentle vortexing.
Let sit on ice for 10 min. 6. Centrifuge for 4 min at 1,300 × g at 4°C in a swinging bucket rotor and drain liquid by pouring off and inverting onto a paper towel for a few seconds. 7. Resuspend in 1/2 volume of PBS. For unfixed nuclei, skip to Step A11. 8. While gently vortexing, add 16% formaldehyde to 0.1% (e.g., 62 μl to 10 ml) and incubate at room temperature for 2 min.
Note: Light fixation reduces the tendency of cells or nuclei to clump in the 300-wash buffer but can interfere with the binding of some antibodies, reducing yield. 9. Stop cross-linking by adding 1.25 M glycine to twice the molar concentration of formaldehyde (e.g., 600 μl to 10 ml). 10. Centrifuge for 4 min at 1,300 × g at 4°C and drain the liquid by pouring off and inverting onto a paper towel for a few seconds.

Resuspend in Wash buffer to a concentration of ~1 million cells per ml. Check nuclei using
ViCell or a cell counter slide.
12. Nuclei may be slowly frozen by aliquoting 900 μl into cryogenic vials containing 100 μl of DMSO, mixed well, then placed in a Mr. Frosty container filled to the line with isopropanol and placed in a -80°C freezer overnight and stored at -80°C long term.
Note: We have found that good results are obtained using native or cross-linked cells even after being stored in the freezer compartment of a side-by-side refrigerator for >6 months. (15 min) 1. Resuspend and withdraw enough of the ConA bead slurry, ensuring that there will be 3.5 μl for each final sample of up to ~50,000 mammalian cells, which yield ≥50% K562 nuclei using this protocol. Transfer the ConA bead slurry into 1 ml of Binding buffer in a 1.5 ml tube.

B. Prepare Concanavalin A-coated beads
Note: This protocol has been used for up to 16 samples (60 µl beads) in 1 ml or 32 samples (120 µl beads) in 2 ml Binding buffer (in a 2 ml tube).  2. Transfer the thawed nuclei suspension in aliquots of no more than ~50,000 starting mammalian cells to each thin-wall 0.5 ml PCR tube and mix with 3.5 µl ConA beads. Attach to the Tube rotator and rotate at room temperature for 10 min.  b. The secondary antibody step is required for CUT&Tag to increase the number of Protein A binding sites for each bound antibody. We have found that without the secondary antibody, the efficiency is very low.
3. Place the tubes on a rotator and rotate at room temperature for 0.5-1 h. 4. After a quick spin (< 500 × g or just enough to remove the liquid from the sides of the tube), place the tubes on the magnet stand to clear and remove and discard the supernatant with two successive draws, using a 20 µl tip with the pipettor set for maximum volume.

5.
With the tubes still on the magnet stand, carefully add 500 µl of Wash buffer. The surface tension will cause the beads to slide up along the side of the tube closest to the magnet. 3. After a quick spin (<500 × g), place the tubes on a rotator at room temperature for 1-2 h. 4. After incubating in the rotator, perform a quick spin and place the tubes in the magnet stand.
5. Carefully remove the supernatant using a 20 µl pipettor twice to avoid disturbing the beads. 6. With the tubes still on the magnet stand, add 500 µl of the 300-wash buffer.
7. Slowly withdraw 470 µl with a 1 ml pipette tip without disturbing the beads as in Step D6. 10 www.bio-protocol.org/e4043  Note: Indexed primers are described by Buenrostro et al. (2015). We do not recommend Nextera or NEB primers which might not anneal efficiently using this PCR protocol.
2. Add 25 µl NEBnext (non-hot start), vortex to mix, and perform a quick spin. Place the tubes immediately in the thermocycler and proceed immediately with the PCR. 11 www.bio-protocol.org/e4043   Tagmentation was performed for 20 min at 37°C in CUTAC-hex buffer. Representative tracks for these samples are shown in Figure 4A.

Data analysis
1. Align paired-end reads to hg19 using Bowtie2 version 2.3.4.3 with options: --end-to-end --verysensitive --no-unal --no-mixed --no-discordant --phred33 -I 10 -X 700. For mapping E. coli carryover fragments, we also use the --no-overlap --no-dovetail options to avoid possible crossmapping of the experimental genome to that of the carry-over E. coli DNA that is used for calibration. Tracks are made as bedgraph files of normalized counts, which are the fraction of 13 www.bio-protocol.org/e4043 total counts at each basepair scaled by the size of the hg19 genome.
Note: To calibrate samples in a series for samples done in parallel using the same antibody, we use counts of E. coli fragments carried over with the pA-Tn5, as for an ordinary spike-in. Our sample script in Github can be used to calibrate based on either a spike-in or E. coli carry-over DNA.
2. Our CUT&Tag Data Processing and Analysis Tutorial provides step-by-step guidance for mapping and analysis of CUT&Tag sequencing data. Most data analysis tools used for ChIPseq data, such as bedtools, Picard, and deepTools, can be used on CUT&Tag data (Figures 3-5). Analysis tools designed specifically for CUT&RUN/Tag data include the SEACR peak caller, also available as a public web server, and CUT&RUNTools.