ABSTRACT

The Assay for Transposase Accessible Chromatin by sequencing (ATAC-seq) is becoming popular in the neuroscience field where chromatin regulation is thought to be involved in neurodevelopment, activity-dependent gene regulation, hormonal and environmental responses, and pathophysiology of neuropsychiatric disorders. The advantages of using ATAC-seq include a small amount of material needed, fast protocol, and the ability to capture a range of gene regulatory elements with a single assay. With increasing interest in chromatin research, it is an imperative to have feasible, reliable assays that are compatible with a range of neuroscience study designs. Here we tested three protocols for neuronal chromatin accessibility analysis, including a varying brain tissue freezing method followed by fluorescence-activated nuclei sorting (FANS) and ATAC-seq. Our study shows that the cryopreservation method impacts the number of open chromatin regions identified from frozen brain tissue using ATAC-seq. However, we show that all protocols generate consistent and robust data and enable the identification of functional regulatory elements in neuronal cells. Our study implies that the broad biological interpretation of chromatin accessibility data is not significantly affected by the freezing condition. We also reveal additional challenges of doing chromatin analysis on post-mortem human brain tissue. Overall, ATAC-seq coupled with FANS is a powerful method to capture cell-type-specific chromatin accessibility information in mouse and human brain. Our study provides alternative brain preservation methods that generate high-quality ATAC-seq data while fitting in different study designs, and further encourages the use of this method to uncover the role of epigenetic (dys)regulation in the brain.

摘要

转座酶可及染色质测序法 (ATAC-seq) 在神经科学领域变得越来越流行，其中染色质调控被认为与神经发育、活动依赖性基因调控、激素和环境反应以及神经精神疾病的病理生理学有关。使用 ATAC-seq 的优势包括所需的材料量少、方案快速，以及能够通过单一分析捕获一系列基因调控元件。随着对染色质研究的兴趣日益增加，必须拥有与一系列神经科学研究设计兼容的可行、可靠的检测方法。在这里，我们测试了三种用于神经元染色质可及性分析的协议，包括一种不同的脑组织冷冻方法，然后是荧光激活核分选 (FANS) 和 ATAC-seq。我们的研究表明，冷冻保存方法会影响使用 ATAC-seq 从冷冻脑组织中识别出的开放染色质区域的数量。然而，我们表明，所有协议都会生成一致且可靠的数据，并能够识别神经元细胞中的功能调节元件。我们的研究表明，染色质可及性数据的广泛生物学解释不受冷冻条件的显着影响。我们还揭示了进行染色质分析的其他挑战 我们的研究表明，染色质可及性数据的广泛生物学解释不受冷冻条件的显着影响。我们还揭示了进行染色质分析的其他挑战 我们的研究表明，染色质可及性数据的广泛生物学解释不受冷冻条件的显着影响。我们还揭示了进行染色质分析的其他挑战死后的人脑组织。总体而言，ATAC-seq 与 FANS 相结合是一种在小鼠和人脑中捕获细胞类型特异性染色质可及性信息的强大方法。我们的研究提供了替代的大脑保存方法，这些方法可以生成高质量的 ATAC-seq 数据，同时适合不同的研究设计，并进一步鼓励使用这种方法来揭示表观遗传（异常）调节在大脑中的作用。