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Nuclear RNA-seq of Drosophila Top3beta-deficient photoreceptor neurons

ABSTRACT: We report the nuclear transcriptome of Drosophila Top3beta-deficient photoreceptor neurons obtained using a tissue-specific approach to purify nuclei and express an siRNA against Top3beta or mCherry for negative control

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE174491 | GEO | 2021/12/31

REPOSITORIES: GEO

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Project description:The method of mRNA tagging was recently developed to isolate mRNA population from muscle tissues in Caenohabditis elegans. This method utilizes a FLAG-tagged poly(A)-binding protein (PABP), which can bind to poly(A)-tail of eukaryotic mRNA. Thus, the mRNA population from specific tissue, in which FLAG- tagged PABP is expressed, can be co-immunoprecipitated with FLAG-tagged PABP using FLAG-specific antibody. To evaluate the applicability of this method to isolate mRNA from specific tissue in Drosophila, we generated Rh1-GLA4/P{UAS-dPABP-FLAG} flies which express a FLAG-tagged PABP in photoreceptor cells R1-R6. We then prepared 4 individual mRNA samples (subset 1: GSM30900-30903)) presumably from photoreceptor cells of these flies by mRNA tagging method. We also prepared 4 individual mRNA samples (subset 2: GSM30832-30835) from whole heads of the Rh1-GAL4/P{UAS-dPABP-FLAG} flies, as well as 6 individual mRNA samples (subset 3: GSM30824-30828, GSM30830) from whole heads of wild type Canton-S flies. The total 14 mRNA samples were used to synthesize target separately and each of the resultant target was used to hybridize with 1 Drosophila Genome Array DrosGenome1 Affymetrix GeneChip. The raw data of the 14 microarray were imported to dChip program (version 1.3). The arrays were normalized to a common baseline array that has the median overall brightness. The expression value for each probe set on each chip was then calculated as model-based expression index (MBEI) by Perfect Match-only model. By comparing the results of subset 1 and subset 2, we found most known photoreceptor-specific genes were indeed enriched by mRNA tagging. Through the comparison of the results of subset 2 and subset 3, we found the mRNAs of most genes known to be involved in fly visual function were underrepresented in subset 2, which could be due to squelching effect of GAL4. By combining the data of subset 1, 2 and 3, we were able to identify 11 new photoreceptor cell-enriched genes.

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Nuclear RNA-seq of Drosophila Top3beta-deficient photoreceptor neurons

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