CUT&RUN for Chromatin Profiling in Caenorhabditis elegans

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 in Caenorhabditis elegans, a model system of great importance to genomic research. Cell dissociation techniques, which are required for this approach, can be challenging in C. elegans due 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 in C. elegans to 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.

The chromatin factors SET-26 and HCF-1 oppose the histone deacetylase HDA-1 in longevity and gene regulation inC. elegans

“…1d), indicating the results were highly reproducible. As previously shown, the SET-26 binding profile obtained by CUT&RUN was highly similar to the previous ChIP-seq profile for SET-26 obtained from our lab 15, 25 . SET-26 and HCF-1 binding were both enriched in promoter regions in the C. elegans genome (Supplementary Fig.…”

“…In experiments in which worms were grown on OP50, embryos were collected from full stock plates as previously described 25 . 3000 embryos were seeded per plate on 15-cm NGM plates containing 1mL of a 25-times concentrated overnight culture of streptamycin-resistant OP50.…”

“…For consistency, in experiments with glp-1 RNAi, control worms laying embryos on L4440 were pre-incubated overnight with L4440 in the same way. Enough embryos were prepared such that 3000 worms were available for each CUT&RUN reaction as previously described 25 . After seeding embryos, plates (either OP50 or RNAi) were incubated at 25 °C for ∼48-52 h until the plates were full primarily of young adults.…”

“…After seeding embryos, plates (either OP50 or RNAi) were incubated at 25 °C for ∼48-52 h until the plates were full primarily of young adults. After reaching the young adult stage, worms were washed off plates and CUT&RUN experiments were performed precisely as previously described by us in detail elsewhere 25 , similarly to other standard CUT&RUN protocols 51, 52 .…”

“…Sequencing libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, cat. No E7645S and E7335S) and amplified using 14 PCR cycles following the manufacturer s instructions with slight modifications as previously described by us in detail elsewhere 25 . Libraries were submitted for 2x32 paired-end sequencing with an Illumina NextSeq 500 machine.…”