Simplified Epigenome Profiling Using Antibody-tethered Tagmentation

Abstract

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.

Keywords: CUT&Tag; Chromatin accessibility; Epigenomic profiling; Histone modifications; RNA polymerase II.

Figure 1.. A home workbench for CUT&Tag.

Photo of the home workbench setup used for all experiments presented using this protocol. A typical experiment begins by mixing cells with activated ConA beads in up to 32 single PCR tubes, with all liquid changes performed on the magnet stand. The only tube transfer is the removal of the purified sequencing-ready libraries from the SPRI beads to fresh tubes for Tapestation analysis and DNA sequencing. The total time from thawing frozen nuclei until elution from SPRI beads is ~8 h.

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.

Figure 3.. Tapestation profiles for a low-cell-number RNAPIIS5P CUTAC experiment.

Tagmentation was performed for 20 min at 37°C in CUTAC-hex buffer. Representative tracks for these samples are shown in Figure 4A.

Figure 4.. Accessible DNA corresponds to binding sites of the initiating form of RNA Polymerase II (RNAPII).

A. Tracks show profiles of the Chromosome 1 histone gene cluster, with 12 small intronless genes expressed at high levels in all dividing cells. Whereas RNAPIIS5P CUT&Tag shows broad enrichment over each of the genes, the CUTAC protocol applied to the RNAPIIS5P epitope, either native (RNAPIIS5P CUTAC-N) or cross-linked (RNAPIIS5P CUTAC-X), yields sharp promoter delineation, better than H3K4me2 CUTAC ± 1,6-hexanediol (turquoise) or the best K562 ATAC-seq datasets (green), all downsampled to 3.2 million mapped fragments. Note the 10-fold difference in scale between RNAPIIS5P CUTAC (0-1500 and K4me2-CUTAC/ATAC (0-150). Similar results were obtained for three mixed-lineage leukemia cell lines (KOPN8, SEM, and RS411) and H1 embryonic stem cells down to ~2,000 cells. No changes were made to the protocol for low cell numbers. Numbers in parentheses are estimated library sizes in millions of mapped paired-end reads. B-D. RNAPIIS5P occupies sites of accessible chromatin in K562 cells. B. Left to right: K4me2 CUT&Tag, K4me2 CUTAC, RNAPIIS5P CUT&Tag, RNAPIIS5P CUTAC, Omni-ATAC, and Fast-ATAC datasets were downsampled to 3.2 million fragments and aligned over ATAC-seq peaks called using MACS2 on data generated by the ENCODE project (ATAC_ENCODE). C. Same as (A) except using only subnucleosome-sized fragments (≤120 bp). CUTAC RNAPIIS5P sites are virtually indistinguishable from high-quality ATAC-seq data, directly demonstrating that ATAC-seq maps sites of the initiation form of RNA Pol II. D. Same as (A) except using only >120 bp fragments. ENCODE ATAC-seq fragments were downsampled to 3.2 million, ChrM (mitochondrial DNA) was removed, and MACS2 was used to call peaks. Heatmaps are centered over ENCODE ATAC-seq peak summits and ordered by occupancy over the 5 kb span displayed. Fast-ATAC is an improved version of ATAC-seq that reduces mitochondrial reads ( Corces et al., 2016 ), and Omni-ATAC is an improved version that additionally improves the signal-to-noise ratio ( Corces et al., 2017 ). ATAC_ENCODE is the current ENCODE standard ( Consortium et al., 2020 ).

Figure 5.. RNAPIIS5P CUTAC shows high sensitivity and specificity.

Mapped fragments from the indicated K562 datasets were sampled, and peaks were called using MACS2. A) Number of peaks (left) and B) fraction of reads in peaks for CUT&Tag (triangles) and CUTAC (squares) profiles for H3K4me2, RNAPIIS5 (initiation form), RNAPIIS2P (elongation form), RNAPIIS2P/S5P, and Omni-ATAC (green). CUTAC for RNAPIIS5P shows the best sensitivity (most peaks at low sampling) and the best signal-to-noise (highest FRiP at all sampling levels). Tagmentation was for 10 min at 37°C in CUTAC-tag buffer.