GSM3178892: Third instar larval testis with attached fat body rep 1.; Drosophila melanogaster; RNA-Seq
We dissected late third instar larvae in PBS at 22°C (Gibco Life Technologies, Carlsbad, CA). Each sample was collected in 350 μl RLT buffer (Qiagen, Cat. No. 79216. Valencia, CA), vortexed two times for 30 sec with a 30 sec interval and quick frozen in a dry ice ethanol bath and stored at -80°C until RNA extraction. We collected testes with the fatbody attached just around the gonads (pool of 10 testes). We collected testes without fatbody by enzymatically removing 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 1.5ml eppendorf tube for 3 min with gentle vortexing by flicking the tube with a forefinger (pool of 15 testes). We collected fatbody alone by dissecting the fatbody around the gonads (pool from 10 testes). We dissociated testes after removing the fatbody either by treating with 0.5% Trypsin (ThermoFisher, Walkersville, MD) and 2 mg/ml Collagenase (Sigma-Aldrich, St. Louis, MO) in PBS for 30 min at 22°C or with 2 mg/ml Papain and 2 mg/ml Collagenase enzymes (pools of 15 testes). To obtain maximum dissociation, testes were teased apart with tungsten needles (Fine Scientific Tools, Foster City, CA) and pipetted several times. We pelleted cells using a table top centrifuge at 0.9 g for 5 min and resuspended in 350 μl RLT buffer.       We extracted total RNA using RNeasy 96 kit (Qiagen, Cat. No. 1019545. Valencia, CA) and followed the manufacturer's protocol. RNA was quantified using Quant-iT RiboGreen quantification kit (Life Technologies, Cat. No. R11490. Carlsbad, CA). 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, 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). We sequenced stranded 42 multiplexed libraries using a single-ended 50 bp strategy on one lane using a HiSeq Illumina 2500 (Illumina, San Diego, CA).