title: GSM3179828: Third instar larval ovary cleaned of fat body with papain rep 3.; Drosophila melanogaster; RNA-Seq
library_construction_protocol: We selected late third instar larvae that were immobilized, but that had not extended their anterior spiracles. We dissected larvae in PBS at 22°C (Gibco Life Technologies, Carlsbad, CA), and collected ovaries from individuals with Sulforhodamine B dye located only in the mid and hindgut. Ovaries were collected in 350 μl RLT buffer (Qiagen, Cat. No. 79216. Valencia, CA), vortexed two times for 30 sec with a 30 sec interval in between, quick frozen in a dry ice ethanol bath, and stored at -80°C until RNA extraction. We had four different treatment conditions: We collected 15 ovaries with fatbody still attached around the gonads. We collected 30 ovaries without fatbody by enzymatically removing the fatbody by treating with 0.075 mg/ml Papain (Sigma-Aldrich, St. Louis, MO) and 0.075 mg/ml Collagenase enzymes (Sigma-Aldrich, St. Louis, MO) in PBS in 1.5 ml eppendorf tube for 3 min with gentle vortexing by flicking the tube with a forefinger. We collected fatbody only from 10 ovaries by dissecting the fatbody and disposing of the ovaries. We dissociated 30 ovaries after removing the fatbody by treating with 2 mg/ml Papain and 2 mg/ml Collagenase enzymes. We pelleted cells from all treatments using a table top centrifuge at 0.9 g for 5 min and resuspended in 350 μl RLT buffer.       Using the RNeasy 96 kit, we extracted total RNA (Qiagen, Cat. No. 1019545. Valencia, CA) by following the manufacturer's protocol. We used Quant-iT RiboGreen quantification kit (Life Technologies, Cat. No. R11490. Carlsbad, CA) to quantify the RNA. RNA was stored in -80°C until the library preparation. We used 200 ng of total RNA and performed RNA-seq library preparation according to Lee et al (doi:10.1101/032920 and Gene Expression Omnibus GSE73920). Briefly, PolyA+ selection was done using Dynabeads Oligo(dT)25 (Life technologies, Grand Island, NY). Selected mRNA was fragmented, and RT was performed with the second strand synthesis by incorporating dUTPs. We used External RNA control consortium spike-ins pools 78A and 78B (SRM2374, Zook et al., 2012. PMID: 22859977) transcribed from standard reference. We used barcoded adaptors for indexing the libraries (Qiagen, 98 TruSeq v2 kit barcoded adaptors, Valencia, CA), and sequenced stranded 42 multiplexed libraries using a single-ended 50 bp strategy on one lane using a HiSeq Illumina 2500 (Illumina, San Diego, CA).