HiCAR: Analysis of Open Chromatin Associated Long‐range Chromatin Interaction Using Low‐Input Materials

Abstract 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