Drosophila melanogaster late third instar larval testes and gonadal fatbody RNA-seq profiling

In order to study the effect of protease treatment on dissected testes, we performed different strategies of cleaning and dissociation on Drosophila melanogaster w1118 larval testes. We collected testes without protease treatment with fatbody just attached around the gonad, fatbody alone dissected from around the testes, testes without fatbody and testes dissociated by either by Papain or Trypsin and Collagenase cocktail. We prepared poly A+ RNA-Seq libraries and performed transcriptional profiling to generate 50 bp stranded single end reads. Overall design: We collected late third instar larvae and dissected testes in PBS at at 22°C. Testes were pooled (see Extraction Protocol for details) and treated as one of 5 sample types: testes with fatbody just attached around the gonad (TF), fatbody from around the testes (FT), testes with fatbody removed by briefly treating with a papain cocktail (TCP), testes dissociated with a papain cocktail (TDP), and testes dissociated with a trypsin cocktail (TDT). All samples were collected in biological quadruplicate. While pools of testes were collected at the same time, different treatments and replicates were collected on different days and stored at -80ºC until RNA extraction and library preparation. RNA extraction and library preparation were performed simultaneously for all samples in a 96 well plate (see Extraction protocol and Library Construction protocol for details).