Copyright © 2020 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License jove.com October 2020 • 164 • e61086 • Page 1 of 13 Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos Rachel A. Keuls 1 , Ronald Parchem 1 1 Development, Disease Models & Therapeutics Graduate Program, Department of Molecular and Cellular Biology, Department of Neuroscience, Center for Cell and Gene Therapy, Stem Cells and Regenerative Medicine Center, Baylor College of Medicine Corresponding Author Ronald Parchem Ronald.Parchem@bcm.edu Citation Keuls, R.A., Parchem, R. Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos. J. Vis. Exp. (164), e61086, doi:10.3791/61086 (2020). Date Published October 9, 2020 DOI 10.3791/61086 URL jove.com/video/61086 Abstract MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo. Introduction A central question of developmental biology is how a single undifferentiated cell can give rise to an entire organism with numerous complex cell types. During embryogenesis, the developmental potential of cells becomes progressively restricted as the organism develops. One example is the neural crest lineage, which progressively differentiates from a multipotent cell population into various terminal derivatives, such as peripheral neurons, glia, cranial bone, and cartilage. Neural crest cells are specified from the ectoderm during gastrulation and then undergo an epithelial to mesenchymal transition and migrate through the embryo to discrete locations throughout the body where they will terminally differentiate 1 . Decades of work have uncovered a transcriptional gene regulatory network, but far less is known