Single-Molecule Multikilobase-Scale Profiling of Chromatin Accessibility Using m6A-SMAC-Seq and m6A-CpG-GpC-SMAC-Seq.
Single-Molecule Multikilobase-Scale Profiling of Chromatin Accessibility Using m6A-SMAC-Seq and m6A-CpG-GpC-SMAC-Seq.
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DOI：
10.1007/978-1-0716-2140-0_15
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发表时间：
2022
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真核生物中活性顺式调控元件（CREs）的一个显著特征是它们的核小体耗用，因此，酶处理的可及性更高。这一特性已成为许多基于测序的全基因组鉴定和追踪cre在不同生物条件下活性的分析的基础，如dna -seq、ATAC-seq、name -seq等。然而，许多这些测定方法固有的DNA片段化和短读测序平台有限的读取长度，到目前为止还不能同时测量彼此相距较远的cre的染色质可及性状态。标记可接近的DNA与DNA修饰和纳米孔测序的结合使得开发这样的测定成为可能。在这里，我们提供了m6A-SMAC-seq和m6A-CpG-GpC-SMAC-seq变体中进行SMAC-seq测定（单分子长读可访问染色质映射测序）的详细方案，以及数据处理和分析方法，并讨论了使用SMAC-seq数据集的关键实验和分析注意事项。
A hallmark feature of active cis-regulatory elements (CREs) in eukaryotes is their nucleosomal depletion and, accordingly, higher accessibility to enzymatic treatment. This property has been the basis of a number of sequencing-based assays for genome-wide identification and tracking the activity of CREs across different biological conditions, such as DNAse-seq, ATAC-seq, NOMe-seq and others. However, the fragmentation of DNA inherent to many of these assays and the limited read length of short-read sequencing platforms have so far not allowed the simultaneous measurement of the chromatin accessibility state of CREs located distally from each other. The combination of labeling accessible DNA with DNA modifications and nanopore sequencing has made it possible to develop such assays. Here, we provide a detailed protocol for carrying out the SMAC-seq assay (Single-Molecule long-read Accessible Chromatin mapping sequencing), in its m6A-SMAC-seq and m6A-CpG-GpC-SMAC-seq variants, together with methods for data processing and analysis, and discuss key experimental and analytical considerations for working with SMAC-seq datasets.