Isolation and identification of De Novo long noncoding RNAs from mouse myoblasts and embryonic stem cells

Abstract

Long noncoding RNAs (lncRNAs) are a pervasive class of transcripts whose importance and biological relevance are only beginning to be elucidated. LncRNAs have been detected in nearly every cell type and found to be fundamentally involved in many biological processes; however, studies that characterize lncRNA expression during certain periods of development are largely missing. Here, we demonstrate how a pool of potentially relevant lncRNAs can be identified using a RNA-chromatin immunoprecipitation (RNA-ChIP) technique that pulls down sufficient amount of RNA to send for sequencing. In our initial experiment, we attempted to identify lncRNAs bound to the MyoD protein in myoblast cells; however, the lack of immunoprecipitation-compatible highly specific antibodies against MyoD prevented us from pursuing this project. As an alternative, we successfully identified lncRNAs bound to the histone-modifying complex COMPASS, as well as those bound to the master pluripotency factors Oct4 and Sox2 in mouse embryonic stem cells. This study provides a proof-of-principle to identify lncRNAs potentially involved in chromatin regulation of pluripotency.