Computer-Assisted Annotation of Murine Sertoli Cell Small RNA Transcriptome1

ABSTRACT Mammalian genomes encode a large number of small noncoding RNAs (sncRNAs) that play regulatory roles during development and adulthood by affecting gene expression. Several sncRNA species, including microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), endogenous small interfering RNAs (endo-siRNAs), and small nucleolar RNAs (snoRNAs), are abundantly expressed in the testis and required for normal testicular development and spermatogenesis. To evaluate global changes in sncRNA expression, the next-generation sequencing (NGS)-based sncRNA transcriptomic analysis has become routine, because it allows rapid determination of the small RNA transcriptome of a particular testicular cell type. However, annotation of small RNA NGS reads can be challenging due to the volume of reads obtained, which is usually in the millions. Therefore, we developed a computer-assisted sncRNA annotation protocol that could identify not only known sncRNAs but also previously uncharacterized ones. Using this protocol, we annotated NGS reads of a Sertoli cell sncRNA library, and we report to our knowledge the first comprehensive annotation of the sncRNA transcriptome of immature murine Sertoli cells. Moreover, the computer-assisted sncRNA annotation pipeline that we report is applicable for annotating NGS reads derived from other cell types and/or sequencing platforms.

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