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Advances in DNA sequencing are enabling new experimental modalities for studying chromatin. One emerging area is to use high-throughput DNA sequencing to monitor dynamic changes occurring to chromatin. O -Linked N -acetylglucosamine ( O -GlcNAc) is a reversible protein modification found on many chromatin-associated proteins. The mechanisms by which O -GlcNAc regulates gene transcription are of high interest. Here we use DNA precipitation methods to enable monitoring time-dependent turnover of O -GlcNAc modified proteins associated with chromatin. Using an antibody-free chemical reporter strategy to map O -GlcNAc to the genome, we performed time course metabolic feeding experiments with wild-type Drosophila larvae alongside larvae lacking O -GlcNAc hydrolase (OGA), which are accordingly unable to remove O -GlcNAc. Analysis of resulting next-generation DNA sequencing data revealed that O -GlcNAc on chromatin-associated proteins at most genomic loci is processed with a half-life in hours. Notably, loss of OGA only increases this half-life by ∼3-fold. Interestingly, a small set of genomic loci are particularly sensitive to loss of OGA. In addition to these observations and new strategies to permit monitoring turnover of O -GlcNAc on chromatin, we also detail methods for coded blinding of samples alongside new normalization strategies to enable time-resolved, genome-wide analyses using chemical genetic methods. We envision these general methods will be applicable to diverse protein and nucleic acid modifications.

A Chemical Genetic Method for Monitoring Genome-Wide Dynamics of O-GlcNAc Turnover on Chromatin-Associated Proteins