Profiling the epigenome at home

Steven Henikoff; Jorja G. Henikoff

The basic CUT&Tag method is schematized in Figure 1 . Our simplified protocol applies to any chromatin feature for which an antibody is available and should be adaptable to any cell type for which there is a standard nuclei isolation protocol. In brief, native or lightly cross-linked nuclei are prepared and immobilized on magnetic beads. Beads are incubated with a primary antibody followed by incubation with a secondary antibody to increase the number of IgG molecules at each epitope bound by the primary antibody. Beads are washed and incubated with protein A-Tn5 loaded with mosaic-end adapters and washed under stringent conditions. Tn5 is activated by addition of Mg 2+ , a "one-and-done" reaction in that the pA-Tn5 transposome is not active once it integrates its adapters. DNA is released in a small volume of SDS and then mixed with Triton-X100 to neutralize the SDS. Samples are enriched by PCR amplification and a single Solid Phase Reversible Immobilization (SPRI) magnetic bead cleanup step. Up to 48 barcoded libraries from multiple experiments may be pooled for each lane of a 2-lane flow cell, as 3 million mapped paired-end reads are usually sufficient for a genome-wide profile of a histone modification in human cells. CUT&Tag@home is performed on frozen nuclei using non-toxic materials, and has minimal equipment requirements, so that it can be conveniently performed in a utility area on ~1.5 meters of counter space (Figure 2) .