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Sequencing of all RNA species by next generation sequencing of wildtype and GCNAKO Drosophila ovaries

ABSTRACT: Sequencing of all RNA species by next generation sequencing of wildtype and GCNAKO Drosophila ovaries

PROVIDER: PRJNA524361 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR8635330.fastq.gz	Fastqsanger.gz
	SRR8635331.fastq.gz	Fastqsanger.gz
	SRR8635332.fastq.gz	Fastqsanger.gz
	SRR8635333.fastq.gz	Fastqsanger.gz
	SRR8635334.fastq.gz	Fastqsanger.gz

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Sequencing of all RNA species by next generation sequencing of wildtype and GCNAKO Drosophila ovaries

Project description:Purpose: To identify significantly different individual mRNA species or genetic pathways in the GCNAKO mutant sample when compared to the wildtype sample. GCNA is encoded by CG14814 (Flybase ID: FBgn0023515). Method: Total RNA was extracted using the Qiagen miRNEasy kit, followed by on column DNAse digest using the Qiagen DNAse digest kit. We used the miRNEasy kit to ensure collection of small RNA species as well as total RNA. RNA was collected from three independent bological replicates of freshly dissected Drosophila ovaries ( 100 ovaries per replicate), collected from 1-3 day old female Drosophila. Ovaries were flash frozen in liquid nitrogen after dissection to preserve the RNA species and prevent degradation. RNAse free conditions were maintained throughout the extraction protocol. RNA quality was tested by the sequencing core before sequencing the RNA.

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Project description:Belle has been known to be co-localized with piRNA-related proteins at the nuage of germline cells during Drosophila oogenesis. However, its role in piRNA biogenesis remains unclear. To reveal whether Belle is involved in regulating piRNA expression, we performed next-generation sequencing analysis of small non-coding RNAs on ovaries harvested from the wild type (W1118) and trans-heterozygous bel[74407/neo30] mutant. Small RNA-seq experiments were performed on three individual ovary samples with the same genotype. For piRNA expression analysis, we performed mapping of three sets of small RNA sequencing reads for each genotype to previously identified eight distinct piRNA clusters located in four different Drosophila chromosomes (from X to 4). Analysis of the piRNA expression profiling from these piRNA cluster loci indicates that some specific piRNA populations were either upregulated or downregulated in bel mutant ovaries compared with wild-type ovaries. Furthermore, we performed systematic analysis by mapping piRNA sequencing reads to sequences of all identified Drosophila transposable elements (TEs) to classify and measure piRNA reads based on their TE targets. Among 124 TE-classified piRNA populations, 9 and 20 of them were upregulated and downregulated (≥2 folds), respectively, in bel74407/neo30 mutant ovaries compared with those from wild-type ovaries. To examine the effect of the bel[74407/neo30] mutation on the ping-pong cycle for secondary piRNA biogenesis, analysis of the ping-pong signature of piRNAs specifically mapped to the retro-element Burdock was performed. The ping-pong signature for the generation of secondary piRNAs was not significantly altered in bel mutants compared with the wild type. These results, taken together, indicate that Bel is not required for primary and secondary piRNA biogenesis, but it is involved in regulating expression of specific subsets of piRNA populations.

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