A low-input CUT&RUN-qPCR method for histone modification profiling in Daphnia magna embryos

Understanding how organisms translate environmental variation into phenotypic variation is a central question in ecology and evolution. Histone modifications are key epigenetic regulators underlying phenotypic plasticity, enabling the generation of distinct phenotypes from a single genome. Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is a powerful method for profiling histone modifications from low amounts of starting material. Here, we adapted a low-input CUT&RUN-qPCR protocol for embryos of the freshwater crustacean Daphnia magna, an emerging model for studying the molecular mechanisms of phenotypic plasticity. We established a cell dissociation protocol and applied the method to the male-determining gene doublesex1 (dsx1) locus during environmentally induced sex determination. CUT&RUN-qPCR detected male-biased enrichment of the active histone mark H3K4me3 at the dsx1 transcription start sites, whereas the repressive marks H3K9me3 and H3K27me3 showed higher enrichment in females. Using a dsx1 reporter strain in which dsx1-expressing cells are labeled with mCherry, we estimated that mCherry-positive cells comprise approximately 11% of dissociated male embryonic cells. Despite this heterogeneity, sex-biased H3K4me3 enrichment at dsx1 remained detectable from cells derived from as few as 20 embryos, corresponding to on the order of a few thousand target cells per reaction. This benchmark provides a practical guideline for planning CUT&RUN input requirements in heterogeneous and low-input samples. Our study establishes CUT&RUN-qPCR as a practical approach for histone modification profiling in Daphnia and provides a methodological basis for future work on the epigenetic underpinnings of phenotypic plasticity.