Effect of deletion of CPES on gene expression during spermatogenesis in Drosophila testes

To investigate the role of CPES in germ cell differentiation during spermatogenesis in Drosophila testis. We have generated cpes null mutants using ends-out homologus recombination and rescued with Bam-Gal4 and UAS-CPES in cpes mutant background. We then dissected 200 pairs of testes for each of the 3 replicates from wild type, cpes mutant and Rescue (Bam-Gal4 and UAS-CPES) Drosophila males and total RNA was extracted using Trizol and RNA-Clean and concentrater column. About 1ug of RNA in 20ul was submitted for bulk RNAseq using Illumina machine (HWI-ST1276). Overall design: Original RNAseq data was assessed and mapped to Drosophila genome. The readcounts were taken input into DESeq r-package and using the function for variant stabilization transformation (vst) the counts were transformed for further analysis. The flybase IDs were mapped to gene symbols using org.dm.eg.db r-package. The vst ransformed counts file was used as one of the input file for Gene Set Enrichment Analysis (GSEA). Linux platform with GSEA v4.02 was used for anlysis. All the pathway sets were downloaded from http://www.go2msig.org/cgi-bin/prebuilt.cig?taxid=7227, Drosophila melanogaster (Fruit fly- taxon 7227) genes identified by gene symbols. All Ontologies with high quality GO annotations only was further used in the analysis. Individual pathways were saved as .gmt file and used for analysis.