GSE33431
Comprehensive assembly and spatio-temporal analysis of the C. elegans 3' UTRome through isolation and sequencing of 3'UTRs in germline-dereived cell types and somatic tissues.
Our computational analyses of sequencing depth and the discovery rates of sequence elements in the 3'UTR strongly demonstrate that interrogation of the 3'UTRome in specific tissues and cell types across development will greatly expand the identification of new 3'UTR isoforms and the sequence elements therein. Therefore, we will generate and sequence the 3'UTRs from the cell types isolated from the developing germline, mature germ cells, and early embryogenesis. In addition, we will identify the 3'UTRs for the transcripts isolated from the major somatic tissues during late- and post-embryonic development. Based on our sequencing estimates, we anticipate that the tissue-specific interrogation of the 3'UTRome will reveal a far greater diversity of novel 3'UTR isoform expression that is masked in the whole-worm 3'UTRome sequence data. Using the transgenic myo-3::PABP strain, we have generated polyA-captured libraries for late embryos and across the major stages of post-embryonic development for the muscle transcriptome. We propose to generate these polyA-captured libraries for transcripts expressed in the other major tissues across development. PolyA-captured libraries will be generated for deep sequencing following the tissue-specific isolation of mRNAs by PABP pulldowns (the PABP immunoprecipitations will take advantage of a FLAG epitope which is fused to PABP in all of the transgenic strains). We propose to validate the expression of tissue- specific transcripts by qPCR for select transcripts known to be expressed in those particular tissues. In addition, following the strategy we have employed for the large-scale polyA-captured libraries from muscle, we will perform quality checks for 3' end capture by manually sequencing ~60 clones isolated from each library. Once we have obtained the deep sequencing data, we will analyze all of the sequence reads using the bioinformatics pipeline that we established for the whole-worm polyA- captured sequences (Mangone et al., 2010). Overall design: Four samples representing gravid, L2, L3, and L4 developmental stages were analyzed.