GSM6211478: L3_XX_tra_lof_gonad_rep2; Drosophila melanogaster; RNA-Seq
GSM6211478
We followed the Extraction Protocol from GSE115478. Briefly, we dissected larvae in 1XPBS at 22°C (Gibco Life Technologies, Carlsbad, CA) and separated the fatbody from the gonads. In order to separate the fatbody, we treated the gonads with 0.075 mg/ml Papain (Sigma-Aldrich, St. Louis, MO) and 0.075 mg/ml Collagenase enzyme (Sigma-Aldrich, St. Louis, MO) mixed in PBS in a 1.5ml Eppendorf tube for 3 min with gentle vertexing by flicking the tube with a forefinger. We washed the gonads twice with 1X PBS after decanting the floating fatbody cells. We collected each sample in a 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. 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) quantified the RNA and stored it at -80°C until the library preparation. We used 200 ng of total RNA and performed RNA-seq library preparation following 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), 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). We sequenced eight multiplexed libraries using a single-ended 50 bp strategy on one lane using a HiSeq Illumina 2500 (Illumina, San Diego, CA).