Mapping open chromatin regions has emerged as a widely used tool for identifying active regulatory elements in eukaryotes. However, existing approaches, limited by reliance on DNA fragmentation and short-read sequencing, cannot provide information about large-scale chromatin states or reveal coordination between the states of distal regulatory elements. We have developed a method for profiling the accessibility of individual chromatin fibers, a single-molecule long-read accessible chromatin mapping sequencing assay (SMAC-seq), enabling the simultaneous, high-resolution, single-molecule assessment of chromatin states at multikilobase length scales. Our strategy is based on combining the preferential methylation of open chromatin regions by DNA methyltransferases with low sequence specificity, in this case EcoGII, an N⁶-methyladenosine (m⁶A) methyltransferase, and the ability of nanopore sequencing to directly read DNA modifications. We demonstrate that aggregate SMAC-seq signals match bulk-level accessibility measurements, observe single-molecule nucleosome and transcription factor protection footprints, and quantify the correlation between chromatin states of distal genomic elements.

映射开放染色质区域已成为一种广泛使用的工具，用于识别真核生物中的活性调节元件。然而，现有方法受限于 DNA 片段化和短读长测序，无法提供有关大规模染色质状态的信息或揭示远端调控元件状态之间的协调。我们开发了一种分析单个染色质纤维可及性的方法，这是一种单分子长读长可及染色质映射测序分析 (SMAC-seq)，能够同时、高分辨率、单分子评估多千碱基长度的染色质状态秤。我们的策略基于结合 DNA 甲基转移酶对开放染色质区域的优先甲基化与低序列特异性，在本例中为 EcoGII，N ⁶-甲基腺苷（m ⁶ A）甲基转移酶，以及纳米孔测序直接读取DNA修饰的能力。我们证明了聚合 SMAC-seq 信号与批量级可访问性测量相匹配，观察单分子核小体和转录因子保护足迹，并量化远端基因组元件染色质状态之间的相关性。