2018/10/12 by Kami Ahmad · 1 citation
Biochemistry, Genetics and Molecular Biology · Agricultural and Biological Sciences · #CRISPR and Genetic Engineering #Insect symbiosis and bacterial influences #Insect Resistance and Genetics
paper · doi:10.17504/protocols.io.umfeu3n
openalex publication_date 2018/10/12 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/14
We have modified the Cleavage Under Targets and Release Using Nuclease (CUT&RUN) method for epigenomic profiling of histone modifications and chromatin proteins to use dissected Drosophila tissues. In CUT&RUN, cells or tissues are permeabilized and incubated under conditions where a factor-specific antibody can bind at sites of a chromatin protein. This antibody is then used to tether a protein A-micrococcal nuclease fusion protein, which upon activation releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing, and leaving the vast majority of DNA behind in the cellular pellet. CUT&RUN outperforms the most widely used Chromatin Immunoprecipitation (ChIP) protocols in resolution, signal-to-noise, and depth of sequencing required. Previous versions of CUT&RUN have used isolated nuclei, mammalian cultured cells, or yeast spheroplasts. Here we have modified the CUT&RUN protocol to manipulate imaginal discs and tissues dissected from larvae with magnetic beads without isolation of cells or nuclei. About 10 imaginal discs provides high-quality data for a histone modification or for a chromatin factor. A simple spike-in strategy is used for accurate quantitation of experiment yields and library preparation. From larvae to purified DNA, CUT&RUN requires 1-2 days with minimal handling after dissection.