GSM3187820
GSM3187820: Whole female tub dam rep2; Drosophila melanogaster; RNA-Seq
We collected whole males (FBbt:00007004; pool of 3-5 individuals) or whole females (FBbt:00007011; pool of 3-5 individuals) by anesthetizing flies using CO2 and placing them into a 1.5 mL eppendorf tube. Dissections to collect gonads were performed at room temperature in 1X Ringer's solution (111 mM NaCl, 1.88 mM KCl, 64 μM NaH2PO4, 816 μM CaCl2, 2.38 mM NaHCO3). We collected ovaries (FBbt:00004865; pools 8-10 pairs) by removing reproductive tracts using forceps and separating ovaries from the common oviduct (FBbt:00004913) while keeping the lateral oviduct (FBbt:00004912). We collected testes (FBbt:00004928; pools of 8-10 pairs) by removing reproductive tract and separating from the other reproductive tissues. The testes breakpoint from other reproductive tissues varied, so some samples may include the seminal vesicle (FBbt:00004958). We stored dissected tissues in a 1.5 mL eppendorf tube without additional buffer. After collection or dissection we stored all samples at -80 ºC.    Total RNA was isolated using the Qiagen RNeasy kit (Germantown, MD, Cat # 74181 or Cat # 74182) following the spin technology section (Protocol for Isolation of Total RNA from Animal Cells using spin technology, Cat#19504) except all centrifugations were modified to ~4,000 g. We constructed libraries using the Truseq PolyA+ stranded mRNA kit (Illumina, San Diego, CA. Cat. No. RS-122-2101 or RS-122-2102), following the manufacturer's protocol, modified to use half reactions with 200 ng total RNA as input for all samples. For each sample we spiked in External RNA Control Consortium (ERCC) spike-ins pool 78A or 78B (SRM2374, Zook et al., 2012. PMID: 22859977). ERCCs were added to the Fragment-Prime-Finish Mix during the mRNA fragmentation step; we added pool 78A to wells A2-A5, A7-A10, and B2-B11 and pool 78B to wells A1, A6, A11, A12, B1 and B12 (20 pg / sample) (Zook et al., 2012. PMID: 22859977).  We multiplexed libraries using 24 differently barcoded adapters from TruSeq (Illumina, San Diego, CA). Libraries were quantified with Quant-iT PicoGreen (Life Technologies, Carlsbad, CA), and pooled for multiplexed sequencing with an Illumina HiSeq 2500 (Illumina, San Diego, CA.).
GEO
GSM3187820
GEO Accession
GSM3187820