title: GSM7868238: RNA-seq bam,hs-bam no HS rep2; Drosophila melanogaster; RNA-Seq
library_construction_protocol: Ovaries were dissected for each sample in cold Dissection buffer (PBS 1X, 0.01% Tween 20, 100 μg/ml cycloheximide) and immediately stored at -80°C after dissection. Frozen samples were homogenised in Polysome extraction buffer (50 mM Tris pH 7.5, 5 mM MgCl2, 150 mM NaCl, 0.5% Triton x-100, 1 mM DTT, 100 μg/ml cycloheximide, protease inhibitor, 25 u/ml Turbo DNAse) using an electrical pestle and then further disrupted by passing 20 times through a 26-gauge needle. The lysate was centrifuged at 20,000g for 10 min at 4°C to pellet tissue debris. Total RNA was isolated by hot phenol-chloroform extraction and quantified using Qubit RNA High Sensitivity Assay Kit (Invitrogen). 1.5 μg of total RNA was used for rRNA depletion by binding of complementary oligos and treatment with RNase H. Libraries were generated using NEBNext®UltraTM Directional RNA Library Prep Kit for Illumina® according to manufacturer9s instructions. Libraries were multiplexed using the NEBNext® Multiplex Oligos for Illumina® and sequenced in paired-end 150 nt-long reads on an Illumina NovaSeq 6000.