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

Genetic Variation at Chromosome 2q13 and Its Potential Influence on Endometriosis Susceptibility Through Effects on the IL-1 Family

Endometriosis is characterized by the growth of epithelial and stromal cells outside the uterine cavity. It has a complex etiology and affects ∼10% of reproductive age women. It is accompanied by a chronic inflammatory response with substantial evidence to indicate genetic susceptibility. The causal genes and their pathways leading to endometriosis, however, are still unknown. Recently, genomewide association studies on endometriosis identified 14 genomic risk loci in women of European and Japanese ancestry. It is becoming increasingly clear that these risk regions are intergenic and thus contribute to disease susceptibility through regulatory mechanisms, most likely mediated through regulation of genes within a restricted distance from the risk variants. One endometriosis risk locus has been detected at chromosome 2q13 within an inflammatory-rich region of gene transcripts and thus may play a role in the inflammation component of the disease. We carried out detailed analysis of the genomic region 250 kb on either side of sentinel SNP rs10167914 and identified 21 transcripts which contained 6 interleukin (IL)-1 family genes, 3 previously reported coding genes that have a relationship to inflammation, 4 novel coding, or pseudogenes, and 8 noncoding RNA transcripts. Through an extensive literature search, we examined the roles these genes and their resultant proteins play in endometriosis pathogenesis. The results suggest alteration in the expression the IL-1 family transcripts either alone or as a complex milieu could have a significant influence on endometriosis and should be prioritized for future study on the implications of inflammation on endometriotic lesions