CUT&Tag-direct for whole cells with CUTAC v2
Résumé
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 DNAin situ.We have since adapted CUT&Tag to also map chromatin accessibility by simply modifying thetransposaseactivation conditions when using histone H3K4me2, H3K4me3 or Serine-5-phosphorylated RNA Polymerase II antibodies. Using these antibodies, tagmentation of accessible DNA sites is redirected 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 tubeswith non-toxic reagents using inexpensive equipment, making our simplified strategy for simultaneous chromatin profiling and accessibility mapping suitable for the lab, home workbench or classroom. New with this version are simple modifications to make the one-tube CUT&Tag-direct protocol suitable for whole cells, while providing data quality comparable to that for Bench-top CUT&Tag.
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