Tissue and cell identity relies heavily on the 3' untranslated region (3'UTR) of mRNAs, which contain several regulatory elements required for proper gene expression. Mechanisms including alternative polyadenylation (APA), which produces distinct 3'UTR isoforms, and downregulation through microRNAs (miRNAs), are essential in establishing tissue identity through modulating gene expression. The networks formed between these distinct mechanisms spin a complex web around tissue and cell identity, which are poorly understood in metazoans. Previous experiments in our lab utilized an immunoprecipitation-based approach which identified APA allows mRNA transcripts to evade miRNA targeting in a tissue-specific manner in the model organism C. elegans. In addition, we identified miRNA targets in the intestine and body muscle tissues, but this approach is unable to identify specific miRNA populations, which are essential pieces in the post-transcriptional regulation puzzle. Identifying tissue-specific miRNA populations will provide a better understanding of how gene regulation modulates identity. With the ultimate goal of producing a comprehensive tissue-specific miRNAome in C. elegans, we developed a novel approach in which RNA is isolated from tissue-specific nuclei using FACS sorting then sequenced. The miRNAs identified with this method are validated using a second unbiased RT-qPCR -based approach. To develop strains expressing fluorescent tissue-specific nuclei, the mCherry fluorochrome was fused to the C. elegans histone H2B ortholog,
his-58. Six worm strains were prepared expressing this construct specifically in the intestine (
ges-1p), body muscle (
myo-3p), hypodermis (
dpy-7p), seam cells (
grd-10p), and excitatory (
nmr-1p) and GABAergic neurons (
unc-47p). Briefly, the worms are homogenized in a tissue grinder with a clearance slightly wider than the diameter of the nuclei, then the lysate is sequentially filtered and centrifuged before FACS isolation. Finally, RNA is isolated and the library is prepped with the Nextera XT kit. Our initial results support the validity of this methodology which will be over-imposed to tissue-specific miRNA targets and the tissue-specific 3'UTRome datasets available in our lab. This will ultimately provide the first comprehensive tissue-specific miRNA and 3'UTR Interactome in a living organism.