Laboratory for Epigenome Inheritance, RIKEN Center for Integrative Medical Sciences, Yokohama, Kanagawa, Japan.
Tokyo Metropolitan University, Hachioji, Tokyo, Japan.
Methods Mol Biol. 2023;2577:83-92. doi: 10.1007/978-1-0716-2724-2_6.
Cleavage Under Target & Release Using Nuclease (CUT&RUN) enables the detection of DNA regions that are bound by a protein of interest. This method is suitable for low-input materials because of the absence of an immunoprecipitation step. However, it sometimes fails when applying it to fragile cells, such as mouse oocytes. Here we describe our low-input CUT&RUN protocol optimized for mouse oocyte and preimplantation embryo samples in which the primary antibody and protein A-MNase binding steps are completed before the cells are bound to Concanavalin A-coated magnetic beads. This modification prevents crush of oocytes and early embryos and unwanted loss of chromatin during CUT&RUN procedures.
利用核酸酶进行靶向切割和释放（CUT&RUN）可检测与目标蛋白结合的 DNA 区域。该方法适用于低投入材料，因为它没有免疫沉淀步骤。然而，当应用于脆弱的细胞（如小鼠卵母细胞）时，该方法有时会失败。在这里，我们描述了优化后的用于小鼠卵母细胞和植入前胚胎样品的低投入 CUT&RUN 方案，其中在细胞与 Concanavalin A 包被的磁珠结合之前，已完成了一抗和蛋白 A-MNase 结合步骤。该改进可防止卵母细胞和早期胚胎的压碎以及 CUT&RUN 过程中染色质的不必要丢失。
