br Introduction Hematopoietic stem cells HSCs are a rare sub

Introduction Hematopoietic stem (-)-JQ1 (HSCs) are a rare subset of cells that possess the abilities of self-renewal and multipotency (Bryder et al., 2006; Gazit et al., 2008). By virtue of these attributes, HSCs can regenerate blood and immune cells throughout life. Prospective isolation of HSCs using defined surface markers enables advanced research (Kiel et al., 2005; Osawa et al., 1996; Spangrude et al., 1988). However, our understanding of adult stem cells is incomplete, as is our ability to utilize them clinically. HSCs enable bone marrow transplantation, as they can naturally reconstitute the blood and immune systems of recipients, thus, saving tens of thousands of patients every year (Copelan, 2006; Thomas, 2005). Nevertheless, their transplantation is still limited because of the need to have a matched donor, without manipulation of the cells, further attesting to our incomplete understanding. Better knowledge of HSCs is essential for the field of adult stem cell research and to extend the application of HSCs in regenerative medicine beyond current practice. The scientific understanding of stem cell functions advanced with molecular biology (Orkin and Zon, 2008; Rossi et al., 2012). Genetic studies have made seminal discoveries, finding the mutations in genes that are associated with hematopoietic diseases (Rosenbauer and Tenen, 2007). Such mutated genes revealed developmental, immune, and cellular processes. The development of microarray techniques has enabled the quantification of whole transcriptomes, revealing the expression of virtually all genes in their respective tissues. Transcriptome data obtained from highly purified HSCs made it possible to identify HSC-specific genes and map networks of genes (Gazit et al., 2013). We used transcriptome data to identify HSC genes that are capable of direct reprogramming blood cells into an induced HSC state (Riddell et al., 2014). However, most of the genes are transcribed into several mature mRNA by alternative splicing (AS), whereas microarray analysis is typically performed at the coarse level describing genes as discrete units. We bypassed this problem by cloning HSC factors for reprogramming directly from the cDNA of HSC (Riddell et al., 2014) but noted that the data lacked the resolution of isoform expression of each gene. High-resolution mapping of the expression of isoforms throughout the HSC transcriptome is needed for better understanding of the specific genes of interest. Fortunately, the emerging technique of RNA-sequencing (RNA-seq) can potentially identify and quantify all of the expressed isoforms simultaneously. Nevertheless, delineating the data into a complete AS map remains a challenge. The splicing process increases transcriptome complexity: instead of having only one product per gene, there can be multiple distinguishable isoforms. Indeed, of 25,000 protein-coding genes, there are at least 10-fold more isoforms (Nilsen and Graveley, 2010). Isoforms include alternative splice-donor and/or splice-acceptor sites that change the length of each exon, or the inclusion/exclusion of an exon. If such an exon encodes for an activation domain, the isoform might invert the gene\'s function as the activation-deficient protein keeps its interaction and becomes an inhibitor. Knowing which isoforms are expressed is of paramount importance, since different tissues may express different isoforms of the same gene. Splicing had been recently mapped in human HSCs (Sun et al., 2014), suggesting significant roles for the maintenance and differentiation of stem cells. Hence, a map of the whole-transcriptome splicing of mouse HSCs is necessary to provide high-resolution data for future studies of these cells and advance the field of adult stem cell research. Following the identification of embryonic stem cell (ESC)-specific AS (Gabut et al., 2011), their regulation had been studied, leading to the identification of muscleblind-like proteins as general negative regulators of stem cell-specific splicing in human cells (Han et al., 2013). Recently, unique AS in pluripotent stem cells has been discovered to be conserved in worms just as in humans (Solana et al., 2016), highlighting its fundamental role. These pioneering studies increased the interest in studying AS and regulation of ESCs, and reprogramming (Aaronson and Meshorer, 2013). Importantly, AS is implicated in many diseases after specific mutations that do not affect the coding sequence were discovered to cause aberrant splicing that can frequently disrupt gene function (Xiong et al., 2015). Clinical interest in aberrant splicing aided the discovery of mutations in splicing factors that drive hematopoietic malignancies (Steensma, 2012). This unstudied dimension of transcriptome complexity is yet to be studied in somatic stem cells.