Background RNA sequencing (RNA-Seq) in its varied forms has become an indispensable tool for analyzing differential gene expression and thus characterization of specific tissues. Aiming to understand the brain barriers genetic signature, RNA seq has also been introduced in brain barriers research. This has led to availability of both, bulk and single-cell RNA-Seq datasets over the last few years. If appropriately performed, the RNA-Seq studies provide powerful datasets that allow for significant deepening of knowledge on the molecular mechanisms that establish the brain barriers. However, RNA-Seq studies comprise complex workflows that require to consider many options and variables before, during and after the proper sequencing process. Main body In the current manuscript, we build on the interdisciplinary experience of the European PhD Training Network BtRAIN ( https://www.btrain-2020.eu/ ) where bioinformaticians and brain barriers researchers collaborated to analyze and establish RNA-Seq datasets on vertebrate brain barriers. The obstacles BtRAIN has identified in this process have been integrated into the present manuscript. It provides guidelines along the entire workflow of brain barriers RNA-Seq studies starting from the overall experimental design to interpretation of results. Focusing on the vertebrate endothelial blood–brain barrier (BBB) and epithelial blood-cerebrospinal-fluid barrier (BCSFB) of the choroid plexus, we provide a step-by-step description of the workflow, highlighting the decisions to be made at each step of the workflow and explaining the strengths and weaknesses of individual choices made. Finally, we propose recommendations for accurate data interpretation and on the information to be included into a publication to ensure appropriate accessibility of the data and reproducibility of the observations by the scientific community. Conclusion Next generation transcriptomic profiling of the brain barriers provides a novel resource for understanding the development, function and pathology of these barrier cells, which is essential for understanding CNS homeostasis and disease. Continuous advancement and sophistication of RNA-Seq will require interdisciplinary approaches between brain barrier researchers and bioinformaticians as successfully performed in BtRAIN. The present guidelines are built on the BtRAIN interdisciplinary experience and aim to facilitate collaboration of brain barriers researchers with bioinformaticians to advance RNA-Seq study design in the brain barriers community.

中文翻译：

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推进脑屏障 RNA 测序：从实验设计到发表的指南

各种形式的背景 RNA 测序 (RNA-Seq) 已成为分析差异基因表达并因此表征特定组织的不可或缺的工具。为了了解脑屏障遗传特征，RNA seq 也被引入脑屏障研究。在过去的几年里，这导致了批量和单细胞 RNA-Seq 数据集的可用性。如果执行得当，RNA-Seq 研究提供了强大的数据集，可以显着加深对建立脑屏障的分子机制的了解。然而，RNA-Seq 研究包含复杂的工作流程，需要在正确的测序过程之前、期间和之后考虑许多选项和变量。正文 在目前的手稿中，我们以欧洲博士培训网络 BtRAIN (https://www.btrain-2020.eu/) 的跨学科经验为基础，生物信息学家和脑屏障研究人员在该网络中合作分析和建立关于脊椎动物脑屏障的 RNA-Seq 数据集。BtRAIN 在此过程中发现的障碍已纳入本手稿。它提供了从整体实验设计到结果解释的脑屏障 RNA-Seq 研究整个工作流程的指南。以脉络丛的脊椎动物内皮血脑屏障 (BBB) 和上皮血脑脊液屏障 (BCSFB) 为重点，我们提供了工作流程的分步描述，强调在工作流程的每个步骤中要做出的决定，并解释所做出的个人选择的优势和劣势。最后，我们就准确的数据解释和出版物中包含的信息提出建议，以确保科学界对数据的适当可访问性和观察结果的可重复性。结论 脑屏障的下一代转录组分析为了解这些屏障细胞的发育、功能和病理学提供了新的资源，这对于了解 CNS 稳态和疾病至关重要。RNA-Seq 的持续进步和复杂性将需要脑屏障研究人员和生物信息学家之间的跨学科方法，正如在 BtRAIN 中成功执行的那样。