PolyA+ RNA-Seq profiling of Drosophila melanogaster late third instar larval ovary, testis and sex transformed gonads as a result of tra and dsx perturbation.

We designed this experiment to investigate the transcriptional changes in gonads as a result of sex transformation. Here we performed transcriptional profiling of the ovary transformed into testis from the tra loss of function (XX_tra_lof), testis transformed into ovary from the tra gain of function (XY_tra_gof) and ovary transformed into testis in dsxM gain of function (XX_DsxM_gof/lof) Drosophila melanogaster third instar larvae in biological quadruplicates. In addition, as controls we sequenced ovaries and testes from the female and male wildtype larvae respectively. We constructed polyA+ libraries of the gonads, cleaned off the fatbody and performed 50 bp, stranded single-end RNA-Seq. We dissected late third instar larvae and pooled (see Extraction protocol for details) 25 ovaries, 15 testes, 25 ovaries transformed into testes from the tra loss of function (XX_tra_lof), 25 testes transformed into ovaries from the tra gain of function (XY_tra_gof) and 25 ovaries transformed into testes from dsxM gain of function (XX_DsxM_gof/lof) larvae in biological quadruplicates. Within a sample type, pools of gonads were collected at the same time, but different replicates and genotypes were collected on different days. We extracted RNA from the samples stored at and constructed libraries simultaneously for all samples in a 96 well plate.