Analysis of Caenorhabditis elegans intestinal gene expression and alternative polyadenylation using fluorescence-activated nuclei sorting (FANS) and 3' end deep sequencing (3'end-seq)

Caenorhabditis elegans is a major eukaryotic experimental system employed to unravel a broad range of cellular and biological processes. Despite the many advantages of C. elegans, biochemical approaches to study tissue-specific gene expression in postembryonic stages are challenging. Here we report a novel experimental approach that enables the efficient determination of tissue-enriched transcriptomes by rapidly releasing nuclei from major tissues of postembryonic animals followed by fluorescence-activated nuclei sorting (FANS). Furthermore, we developed and applied a deep sequencing method, named 3'end-seq, which is designed to examine gene expression and identify 3' ends of transcripts using a small quantity of input RNA. In agreement with intestinal specific gene expression, promoter elements of highly expressed genes are enriched for GATA elements and their functional properties are associated with processes that are characteristic for the intestine. In addition, we systematically mapped pre-mRNA cleavage and polyadenylation sites, or polyA sites, including >3,000 sites that have previously not been identified. The analysis of nuclear mRNA revealed widespread alternative polyA site use in intestinally expressed genes. We describe several novel approaches that will be of significance to the analysis of tissue specific gene expression using small quantity RNA samples from C. elegans and beyond. 3'end-seq of transcriptomes for input and sorted nuclei