Advancing brain barriers RNA sequencing: guidelines from experimental design to publication

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

Serum-free growth of normal and tumor mouse mammary epithelial cells in primary culture.

Freshly isolated normal and tumor mouse mammary epithelial cells embedded within a collagen gel matrix undergo sustained growth when cultured for as long as 3 wk in a serum-free medium composed of a 1:1 (vol/vol) mixture of Hepesbuffered Ham's F12 and Dulbecco's modified Eagle's medium supplemented with insulin, epidermal growth factor (EGF), transferrin, bovine serum albumin fraction V, and cholera toxin. Of these additives, only insulin, EGF, and albumin are required for the growth of most normal cells. Albumin is not always an absolute requirement for growth but greatly enhances it. Lithium has been found to stimulate the growth of normal cells and can replace EGF. The collagen matrix culture system allows sustained growth of primary cultures of both normal and neoplastic mammary epithelium in serum-free conditions. This serum-free system will be useful in identifying and investigating the role of hormones, growth factors, and nutritional factors in regulating the growth of mammary epithelial cells