Department of Cardiac Development and Remodeling, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany.
German Centre for Cardiovascular Research (DZHK), Partner site Rhein-Main, Frankfurt am Main, Germany.
Methods Mol Biol. 2023;2640:397-412. doi: 10.1007/978-1-0716-3036-5_27.
Actively transcribed genes harbor cis-regulatory modules with comparatively low nucleosome occupancy and few high-order structures (="open chromatin"), whereas non-transcribed genes are characterized by high nucleosome density and extensive interactions between nucleosomes (="closed chromatin"), preventing transcription factor binding. Knowledge about chromatin accessibility is crucial to understand gene regulatory networks determining cellular decisions. Several techniques are available to map chromatin accessibility, among which the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) is one of the most popular. ATAC-seq is based on a straightforward and robust protocol but requires adjustments for different cell types. Here, we describe an optimized protocol for ATAC-seq of freshly isolated murine muscle stem cells. We provide details for the isolation of MuSC, tagmentation, library amplification, double-sided SPRI bead cleanup, and library quality assessment and give recommendations for sequencing parameters and downstream analysis. The protocol should facilitate generation of high-quality data sets of chromatin accessibility in MuSCs, even for newcomers to the field.
活性转录基因含有具有相对较低核小体占有率和较少高级结构（“开放染色质”）的顺式调控模块，而非转录基因的特征是核小体密度高，核小体之间广泛相互作用（“封闭染色质”），从而阻止转录因子结合。了解染色质可及性对于理解决定细胞决策的基因调控网络至关重要。有几种技术可用于绘制染色质可及性图谱，其中使用测序的转座酶可及染色质分析（ATAC-seq）是最受欢迎的技术之一。ATAC-seq 基于简单而稳健的方案，但需要针对不同的细胞类型进行调整。在这里，我们描述了一种优化的用于新鲜分离的鼠肌肉干细胞的 ATAC-seq 方案。我们提供了 MuSC 分离、标签酶切、文库扩增、双面 SPRI 珠清洗以及文库质量评估的详细信息，并为测序参数和下游分析提供了建议。该方案应有助于生成 MuSC 中染色质可及性的高质量数据集，即使对于该领域的新手也是如此。
