GSM5388252: Adult_Testes_BM24_Sterile_translocation_rep4; Drosophila melanogaster; ssRNA-seq
We dissected L3 larvae and adults in 1XPBS at 22°C (Gibco Life Technologies, Carlsbad, CA). We collected L3 testes without fatbody by enzymatically removing fatbody following the Extraction Protocol from GSE115478. Briefly, by treating the testes with 0.075 mg/ml Papain (Sigma-Aldrich, St. Louis, MO) and 0.075 mg/ml Collagenase enzyme (Sigma-Aldrich, St. Louis, MO) cocktail in PBS in 1.5ml Eppendorf tube for 3 min with gentle vortexing by flicking the tube with a forefinger, we separated the fatbody from testes. Once we decanted the floating fatbody cells, washed the testes 2X with 1XPBS. We separated the adult testis from seminal vesicle. We collected each sample in 350 μl RLT buffer (Qiagen, Cat. No. 79216. Valencia, CA), vortexed two times for 30 sec with a 30 sec interval and quickly frozen in a dry ice ethanol bath and stored at -80°C until RNA extraction. 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 350 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), fragmented the selected mRNA, performed RT 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 eight multiplexed libraries using a single-ended 50 bp strategy on one lane using a HiSeq Illumina 2500 (Illumina, San Diego, CA).