Genome-Wide Profiling of Histone Modifications in Fission Yeast Using CUT&Tag

Torres-Garcia, S., Huang, Y., Dewornu, F.S., Tong, P., Yeboah, R., Allshire, R., and Shukla, M.

By Dipika Mishra, Akiyoshi Lab

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is one of the most widely used tools in molecular biology to probe genome wide variation of chromatin proteins and their post-translational modifications. The basic protocol involves crosslinking proteins to DNA using formaldehyde followed by fragmentation of cross-linked DNA. In the next step, the DNA interacting protein is immunoprecipitated using specific antibody, the crosslinks are reversed by heat and the associated DNA is then analyzed. Despite recent efforts to optimize ChIP-Seq, current protocols are time-consuming, tedious and involves a large amount of sample. To overcome these limitations, CUT&Tag (Cleavage Under Target and Tagmentation) has been developed. In this method, after permeabilization of the cells, a chromatin protein is bound by a specific antibody, which can then tether a Tn5 transposase fusion protein. In the next step, a targeted tagmentation reaction is used which results in cleavage and adaptor tagging of chromatin regions enriched for the protein or histone modification. The amplification and sequencing of tagged DNA fragments results in high resolution mapping of epigenetic features of the genome. 

Researchers in Allshire lab have developed an adapted version of CUT&Tag protocol to map the histone modifications in fission yeast. In this method, cells are cross-linked followed by spheroplasting, immobilization on ConA beads and permeabilization. Following this, a specific antibody is used that can then recruit the Tn5 transposase fusion protein. This adapted version of the protocol uses only 100ml culture or approximately 10⁶ Schizosaccharomyces pombe cells to generate a high-resolution map of the genome.