C. elegans: total mRNA vs. AWA-specifc mRNA
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PROVIDER: PRJNA448452 | ENA |
REPOSITORIES: ENA
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Project description:Transcriptional profiling of AWA neurons comparing total mRNA of C. elegans The aforementioned results provided us a clear indication that a GPCR expressed in AWA would likely be used for pheromone perception. To get a male AWA-specific expression profile, we adopted a poly-A-binding protein (PABP) mRNA pull-down approach to isolate cell-specific genes (Kunitomo et al., 2005) and co-transformed into the worms a sex determination gene, fem-3, to masculinize its nervous system. Ectopic expression of fem-3 in all neurons can change the entire nervous system to adopt a male identity (Ahringer & Kimble, 1991; Rosenquist & Kimble, 1988). We established the male AWA single-cell transcriptional profile by pooling mRNA from AWA with ectopic expression of PABP there driven by an AWA-specific promoter from odr-7. This PABP fused with an epitope protein tag (FLAG tag) is then extracted using the corresponding antibody, while AWA enriched transcripts with poly-A tail, including the GPCRs, were pulled down together and subjected to microarray analysis. The results confirmed the existence of dyp-13 (hypodermal gene), myo-3 (muscle gene) and odr-10 (AWA-specific over-expression gene) in total RNA samples while odr-10 was found to be over-expressed in AWA enriched sample, where dpy-13 and myo-3 were depleted. The pull-down RNA sample was labeled with cyanine 5 (which is excited by a 532nm laser) and the total RNA sample was labeled with cyanine 3 (which is excited by a 633nm laser). Comparison between transcriptome profiles revealed key differences between whole worm and male AWA neurons, which allows identification of male AWA-enriched transcripts including those encoding GPCRs.
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Project description:To discover new miRNA targets, we generated a C. elegans transgenic line expressing a functional N-terminally Tandem Affinity Purification (TAP) tagged ALG-1 protein (C. elegans strain WS4303). We crossed the TAP::ALG-1 transgene into the mir-58(n4640) mutant background to generate the strain WS5041. For simplicity, we will hereafter term the TAP::ALG-1 transgenic animals as “wild type” and the transgenic WS5041 animals as “mir-58”. In addition to immunopurifying the TAP::ALG-1 and associated RNAs from these strains, we also compared total mRNA levels between these two strains. Total RNA was isolated from the same WS4303 and WS5041 total extracts that was further used for the TAP::ALG-1 RIP. Three independent biological replicates were analyzed. Long-oligo whole-genome C. elegans arrays, produced by the Genome Sequencing Center at Washington University in St. Louis (http://genome.wustl.edu/genome/celegans/microarray/ma_gen_info.cgi), were used for these experiments. A total of 10 µg of total RNA was used for cDNA synthesis.
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