Abstract
Cleavage under targets and release using nuclease (CUT&RUN) is a recently developed chromatin profiling technique that uses a targeted micrococcal nuclease cleavage strategy to obtain high‐resolution binding profiles of protein factors or to map histones with specific post‐translational modifications. Due to its high sensitivity, CUT&RUN allows quality binding profiles to be obtained with only a fraction of the starting material and sequencing depth typically required for other chromatin profiling techniques such as chromatin immunoprecipitation. Although CUT&RUN has been widely adopted in multiple model systems, it has rarely been utilized inCaenorhabditis elegans, a model system of great importance to genomic research. Cell dissociation techniques, which are required for this approach, can be challenging inC. elegansdue to the toughness of the worm's cuticle and the sensitivity of the cells themselves. Here, we describe a robust CUT&RUN protocol for use inC. elegansto determine the genome‐wide localization of protein factors and specific histone marks. With a simple protocol utilizing live, uncrosslinked tissue as the starting material, performing CUT&RUN in worms has the potential to produce physiologically relevant data at a higher resolution than chromatin immunoprecipitation. This protocol involves a simple dissociation step to uniformly permeabilize worms while avoiding sample loss or cell damage, resulting in high‐quality CUT&RUN profiles with as few as 100 worms and detectable signal with as few as 10 worms. This represents a significant advancement over chromatin immunoprecipitation, which typically uses thousands or hundreds of thousands of worms for a single experiment. The protocols presented here provide a detailed description of worm growth, sample preparation, CUT&RUN workflow, library preparation for high‐throughput sequencing, and a basic overview of data analysis, making CUT&RUN simple and accessible for any worm lab. © 2022 Wiley Periodicals LLC.
Basic Protocol 1: Growth and synchronization ofC. elegans
Basic Protocol 2: Worm dissociation, sample preparation, and optimization
Basic Protocol 3: CUT&RUN chromatin profiling
Alternate Protocol: Improving CUT&RUN signal using a secondary antibody
Basic Protocol 4: CUT&RUN library preparation for Illumina high‐throughput sequencing
Basic Protocol 5: Basic data analysis using Linux