Department of Cell Biology, Duke University Medical Center, Durham, North Carolina.
Duke Regeneration Center, Duke University Medical Center, Durham, North Carolina.
Curr Protoc. 2023 Oct;3(10):e899. doi: 10.1002/cpz1.899.
Cis-regulatory elements (cREs) and their long-range interactions are crucial for spatial-temporal gene regulation. While cREs can be characterized as accessible chromatin sequences, comprehensively identifying their spatial interactions remains a challenge. We recently developed a method, HiCAR (Hi-C on Accessible Regulatory DNA), which combines Tn5 transposase and chromatin proximity ligation to analyze open chromatin-anchored interactions in low-input cells. Application of HiCAR in human embryonic stem cells and lymphoblastoid cells reveals high-resolution chromatin contacts with efficiency comparable to in situ Hi-C across various distance ranges. Moreover, HiCAR was successfully applied to 30,000 primary human muscle stem cells, showcasing its potential for analyzing chromatin accessibility and looping in low-input primary cells and clinical samples. Here, we provide a detailed step-by-step protocol to perform the updated HiCAR experiments. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Tn5 Transposase Assembly Basic Protocol 2: HiCAR Library Preparation.
顺式调控元件 (cREs) 及其长程相互作用对于时空基因调控至关重要。虽然 cREs 可以被表征为可及染色质序列，但全面识别它们的空间相互作用仍然是一个挑战。我们最近开发了一种方法，HiCAR（可及调控 DNA 的 Hi-C），它结合了 Tn5 转座酶和染色质邻近连接来分析低输入细胞中开放染色质锚定的相互作用。HiCAR 在人胚胎干细胞和淋巴母细胞中的应用揭示了具有与各种距离范围内的原位 Hi-C 相当的效率的高分辨率染色质接触。此外，HiCAR 成功应用于 30000 个人类肌肉干细胞，展示了其在分析低输入原代细胞和临床样本中的染色质可及性和环化的潜力。在这里，我们提供了一个详细的分步协议来执行更新的 HiCAR 实验。© 2023Wiley Periodicals LLC. 基本方案 1：Tn5 转座酶组装 基本方案 2：HiCAR 文库制备。
