A transcriptomic (RNA-seq) analysis of Drosophila melanogaster adult testes overexpressing microRNA-2b-1

MicroRNAs (miRNAs) are short non-coding single-stranded RNAs with approximately 22 nucleotides in length that negatively regulate the mRNA translation of a target gene. MiR-2b-1 belongs to the largest miR-2 family in Drosophila melanogaster with 8 members and this miRNA family is conserved in invertebrates. miRNAs play key roles in gene regulation, cell proliferation, cell death, cell differentiation and cell developmental homeostasis in multicellular organisms. Its role in various human diseases is continuously being studied. miRNAs also found out to be crucial in maintaining stem cell niche in D. melanogaster gonads. We have identified that ectopic overexpression of miR-2b-1 of D. melanogaster causes testicular bulging (a tumour like phenotype) in 3-5 days old adult flies. Hence, we have performed a transcriptomic (RNA-seq) analysis to understand the role of miR-2b-1 in the development, maintenance, and differentiation of D. melanogaster adult testis stem cells. Data are available from GEO (accession number GSE211399).

RNA-seq Transcriptomics Drosophila melanogaster Testicular bulging Testes miR-2b-1 miRNAs a b s t r a c t MicroRNAs (miRNAs) are short non-coding single-stranded RNAs with approximately 22 nucleotides in length that negatively regulate the mRNA translation of a target gene. MiR-2b-1 belongs to the largest miR-2 family in Drosophila melanogaster with 8 members and this miRNA family is conserved in invertebrates. miRNAs play key roles in gene regulation, cell proliferation, cell death, cell differentiation and cell developmental homeostasis in multicellular organisms. Its role in various human diseases is continuously being studied. miRNAs also found out to be crucial in maintaining stem cell niche in D. melanogaster gonads. We have identified that ectopic overexpression of miR-2b-1 of D. melanogaster causes testicular bulging (a tumour like phenotype) in 3-5 days old adult flies. Hence, we have performed a transcriptomic (RNAseq) analysis to understand the role of miR-2b-1 in the development, maintenance, and differentiation of D. melanogaster adult testis stem cells. Data are available from GEO (accession number GSE211399).

Value of the Data
• Various studies have shown the role of miRNAs in the development and maintenance of stem cells in Drosophila testes. However, this data is the first to report the transcriptomic analysis of testicular bulging in Drosophila melanogaster upon an ectopic overexpression of a miRNA. • This analysis could provide a comprehensive overview of the role of miR-2b-1 in the development of D. melanogaster testicular stem cell niche. • This data could shed light on the crucial role of miRNAs in regulating the genes that are crucial for the stem cell development, maintenance, and differentiation. • The identification of pathways that are regulated by the miR-2b-1, could assist a better understanding of the role of the largest miRNA family (miR-2) in the development of D. melanogaster .

Objective
This data was analyzed to look at the genes and biological processes that are differentially regulated in miR-2b-1 overexpressing bulged testes.

Data Description
Micro RNAs (miRNAs) are short, (approximately 22 nt long) non-coding RNAs that are endogenously regulates genes that are required for various developmental processes [1] . mir-2 is the largest miRNA family in D. melanogaster with 8 members; mir-2a-1, mir-2a-2, mir-2b-1, mir-2b-2, mir-2c, mir-13a, mir-13b-1 and mir-13b-2 [2] . In this study, we found that overexpression of miR-2b-1 alone is sufficient to cause testicular bulging phenotype in D. melanogaster . In order to understand the genes and/or pathways regulated by the miR-2b-1 for the observed phenotype, miR-2b-1 overexpressing 3-5 days old adult male flies were dissected to obtain the testes for RNA extraction and sequencing. RNA sequencing was performed using Illumina Hiseq platform. Six paired-end raw reads were generated, 3 for control ( act5C-GAL4 > OreR ) and 3 for miR-2b-1 overexpression ( act5C-GAL4 > UAS-miR-2b-1 ) respectively. The clean raw reads were mapped using RNA STAR and differential gene expression was performed using edgeR. Galaxy version 22.05 was used to perform these analyses. Differentially expressed genes along with their respective fold change and expression levels as count per million (CPM) are listed in Supplementary 1. The significantly differentially expressed genes are then classified according to the Gene ontology (GO) terms and these data are presented in the Figs. 1 , 2 and 3 .

Total RNA extraction, library construction, and RNA-seq
Approximately 90-100 testes were pooled together for each replicate of RNA extraction using the combination of Trizol reagent (Invitrogen, USA) and RNeasy Mini Kit (Qiagen, Germany) as previously mentioned in Woo et al [3] . Total RNA was used for cDNA library construction following the protocol supplied with the Truseq TM RNA sample prep Kit (Illumina, San Diego, USA). Amplified cDNA fragments were sequenced by Illumina HiSeq TM platform with 2 × 150bp. Raw data generated was trimmed and cleaned by removing low quality reads and removing the adaptor.

Differential expression analysis
Galaxy version 22.05 was used to perform differential gene expression [4] . Cleaned RNA-seq reads were aligned to the reference genome of D. melanogaster by using RNA STAR version 2.7.8a [2] . The genome file, Drosophila_melanogaster.BDGP6.87.gtf was downloaded from Ensembl. To measure gene expression counts featureCounts was used [3] . Differential gene expression was analyzed using edgeR [5] . FDR < 0.05 were set as the threshold for significantly differential expression genes.

GO classification and enrichment
DAVID online tool was used to identify significantly enriched GO terms ( P -value < 0.05) featuring biological process, cellular component, and molecular function [6] .

Ethics Statements
All animal handlings complied with guidelines set forth by the National Institutes of Health for the care and use of laboratory animals, and the protocol of this study followed the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978) and Guide for the Care and Use of Laboratory Animals: Table 4 8th Edition. Only adult male flies were used for data collection.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.